Basal cell carcinoma tumor suppressor gene

ABSTRACT

This invention provides for a tumor suppressor gene inactivation of which is a causal factor in nevoid basal cell carcinoma syndrome and various sporadic basal cell carcinomas. The NBCCS gene is a homologue of the Drosophila patched (ptc) gene.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 60/017,906, filed on May 17, 1996 and U.S. application Ser. No:60/019765, filed on Jun. 14, 1996, both of which are herein incorporatedby reference for all purposes.

BACKGROUND OF THE INVENTION

[0002] This invention pertains to the field of oncology. In particular,this invention pertains to the discovery of a tumor suppressor geneimplicated in the etiology of nevoid basal cell carcinoma syndrome(NBCCS) and various cancers including basal cell carcinomas.

[0003] Many cancers are believed to result from a series of geneticalterations leading to progressive disordering of normal cellular growthmechanisms (Nowell (1976) Science 194:23, Foulds (1958) J. Chronic Dis.8:2). In particular, the deletion or multiplication of copies of wholechromosomes or chromosomal segments, or specific regions of the genomeare common (see, e.g., Smith et al. (1991) Breast Cancer Res. Treat. 18:Suppl. 1: 5-14; van de Vijer & Nusse (1991) Biochim. Biophlys. Acta.1072: 33-50; Sato et al. (1990) Cancer. Res. 50: 7184-7189). Inparticular, the amplification and deletion of DNA sequences containingproto-oncogenes and tumor-suppressor genes, respectively, are frequentlycharacteristic of tumorigenesis. Dutrillaux et al. (1990) Cancer Genet.Cytogenet. 49: 203-217.

[0004] One cancer-related syndrome that appears to have a strong geneticbase is the nevoid basal cell carcinoma syndrome (NBCCS). The nevoidbasal cell carcinoma syndrome, also known as Gorlin syndrome and thebasal cell nevus syndrome, is an autosomal dominant disorder thatpredisposes to both cancer and developmental defects (Gorlin (1995)Dermatologic Clinics 13: 113-125). Its prevalence has been estimated at1 per 56,000, and 1-2% of medulloblastomas and 0.5% of basal cellcarcinomas (BCCs) are attributable to the syndrome (Springate (1986) J.Pediatr. Surg. 21: 908-910; Evans et al. (1991) British J. Cancer. 64:959-961). In addition to basal cell carcinomas (BCCs) andmedulloblastomas, NBCCS patients are also at an increased risk forovarian fibromas, meningiomas, fibrosarcomas, rhabdomyosarcomas, cardiacfibromas and ovarian dermoids (Evans et al. (1991) supra., Evans et al.(1993) J. Med. Genet. 30: 460-464; Gorlin (1995) supra.).

[0005] Non-neoplastic features including, odontogenic keratocysts (whichare most aggressive in the second and third decades of life),pathognomonic dyskeratotic pittina of the hands and feet, andprogressive intracranial calcification (usually evident from the seconddecade) are very common. There is a broad range of skeletal defects(Gorlin (1995) supra.; Shanley et al. (1994) Am. J. Med. Genet. 50:282-290) including rib, vertebral and shoulder anomalies, pectusexcavatum, immobile thumbs and polydactyly. Craniofacial and brainabnormalities include cleft palate, characteristic coarse fades,strabismus, dysgenesis of the corpus callosum macrocephaly and frontalbossing (Gorlin (1995) supra.). Generalized overgrowth (Bale et al.(1991) Am. J. Med. Genet. 40: 206-210) and acromegalic appearance arecommon, but growth hormone and IGF1 levels are not elevated.

[0006] Implications for the affected individual can be severe,predominantly due to the prolific basal cell carcinomas which can numbermore than 500 in a lifetime (Shanley et al. (1994) supra). Expression ofmany features of the syndrome is variable, but the severity tends tobreed true within families (Anderson et al. (1967) Am. J. Hum. Genet.,19: 12-22). This variation between families may reflect specificphenotypic effects of different mutations, modifier genes, orenvironmental factors (sunlight exposure is likely to play a role in theage of onset and incidence of basal cell carcinomas). One third to onehalf of patients have no affected relatives and are presumed to be theproduct of new germ cell mutations (Gorlin (1995) supra.). Unilateraland segmental NBCCS are attributed to somatic mutation in one cell of anearly embryo (Gutierrez and Mora (1986) J. Am. Acad. Dermatol. 15:1023-1029).

[0007] The NBCCS syndrome was mapped to one or more genes at chromosome9q22-31 (Gailani et al. (1992) Cell 69: 111-117; Reis et al. (1992)Lancet 339: 617; Farndon et al. (1992) Lancet 339: 581-2). In addition,it has been demonstrated that the same region is deleted in a highpercentage of basal cell carcinomas and other tumors related to thedisorder (Gailani et al. (1992) supra.) thus suggesting that the NBCCSgene functions as a tumor suppressor. Inactivation of NBCCS gene(s) maybe a necessary if not sufficient event for the development of basal cellcarcinomas (Shanley et al. (1995) Hum. Mol. Genet. 4: 129-133; Gailaniet al. (1996) J. Natl. Canc. Inst. 88: 349-354).

[0008] Since the original mapping of the gene in 1992, linkage studieshave narrowed the NBCCS region to a 4 cM interval between D9S180 andD9S196 (Goldstein et al. (1994) Am. J. Hum. Genet. 54: 765-773; Wickinget al. (1994) Genomics 22: 505-511). Reported recombination involving anunaffected individual tentatively placed the gene proximal to D9S287(Farndon et al. (1994) Genomics 23: 486-489). The 9q22 region, however,is very gene rich and appeared to contain at least two tumor suppressorgenes. In addition, Harshrnan et al. (1995) Hum. Mol. Genet. 4:1259-1266, showed that different methods of identifying cDNAs from agenomic region result in a surprisingly different array of candidategenes. Thus, prior to this invention the specific NBCCS gene wasunknown.

SUMMARY OF THE INVENTION

[0009] This invention provides for a nucleic acid sequence (e.g., acDNA) associated with nevoid basal cell carcinoma syndrome (NBCCS) andwith various cancers including various sporadic basal cell carcinomas(BCCs). The NBCCS gene disclosed herein appears to be a tumor-suppressorgene and is a homologue of the Drosophila patched (ptc) gene. The humanNBCCS gene is therefore also referred to herein as the human PATCHED(PTC) gene.

[0010] Absence, partial inactivation (e.g., through haploinsufficiencyor mutation), complete inactivation, or otherwise altered expression ofthe NBCCS (PTC) gene causes or creates a predisposition to NBCCS and/orto the onset of basal cell carcinomas.

[0011] In one preferred embodiment, this invention provides an isolatedhuman nucleic acid encoding a nevoid basal cell carcinoma syndrome(NBCCS) (PTC) protein, wherein said nucleic acid specificallyhybridizes, under stringent conditions, to a second nucleic acidconsisting of a nucleic acid sequence selected from the group consistingof SEQ ID NO: 1, SEQ ID NO: 58, and SEQ. ID NO: 59, in the presence of ahuman genomic library under stringent conditions. The isolated nucleicacid is at least 30, preferably at least 50, more preferably at least100, and most preferably at least 200 nucleotides in length.

[0012] In another embodiment, the isolated NBCCS nucleic acid has atleast 75 percent sequence identity, preferably at least 85 percent,sequence identity, more preferably at least 90% sequence identity andmost preferably at least 95 percent or even at least 98% sequenceidentity across a window of at least 30 nucleotides, preferably across awindow of at least 50 nucleotides, more preferably across a window of atleast 80 nucleotides, and most preferably across a window of at least100 nucleotides, 200 nucleotides, 500 nucleotides or even the fulllength with the nucleic acid of SEQ ID Nos: 1, 58, or 59.

[0013] In one embodiment, the isolated human NBCCS nucleic acid isamplified from a genomic library using any of the primer pairs providedin Table 2. In another embodiment, the NBCCS nucleic acid is identifiedby specific hybridization with any of the nucleic acids amplified from agenomic library using any of the primer pairs provided in Table 2. In aparticularly preferred embodiment the nucleic acid is a nucleic acidselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 58, andSEQ. ID NO: 59.

[0014] In another embodiment, this invention provides for an isolatedhuman nevoid basal cell carcinoma syndrome (NBCCS) (PTC) nucleic acidsequence, wherein said nucleic acid encodes a polypeptide subsequence ofat least 10 contiguous amino acid residues of the polypeptide encoded bya nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 58, and SEQ. ID NO: 59, or conservative substitutions ofsaid polypeptide subsequence. The isolated human NBCCS nucleic acid ispreferably at least 50, more preferably at least 100, and mostpreferably at least 200, 400, 500, or even 800 residues (amino acids) inlength. In a particularly preferred embodiment, the nucleic acid encodesa polypeptide sequence encoded by a nucleic acid selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 58, and SEQ. ID NO: 59. Even morepreferably, the NBCCS nucleic acid is a nucleic acid selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 58, and SEQ. ID NO: 59.

[0015] In still yet another embodiment, this invention provides anisolated nucleic acid encoding a human nevoid basal cell carcinoma(NBCCS) (PTC) polypeptide comprising at least 10 contiguous amino acidsfrom a polypeptide sequence encoded by a nucleic acid selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 58, and SEQ. ID NO: 59,wherein: said polypeptide, when presented as an antigen, elicits theproduction of an antibody which specifically binds to a polypeptidesequence encoded by a nucleic acid selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 58, and SEQ. ID NO: 59; and said polypeptidedoes not bind to antisera raised against a polypeptide encoded by anucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 58, and SEQ. ID NO: 59 which has been fully immunosorbedwith a polypeptide encoded by a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 58, and SEQ. ID NO: 59.Even more preferably this NBCCS nucleic acid hybridizes to a clone ofthe human PTC gene present in a human genomic library under stringentconditions and even more preferably hybridizes to a nucleic acidselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 58, andSEQ. ID NO: 59.

[0016] The invention also provides isolated nucleic acids that includeone or more mutations compared to a nucleic acid selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 58, and SEQ. ID NO: 59. Themutations can be, for example, missense mutations, nonsense mutations,frameshift mutations, and splicing mutations: Alternatively, themutations can be in regulatory regions that affect expression of theNBCCS gene.

[0017] In another embodiment, this invention provides for vectorsincorporating any of the above-described nucleic acids. The vectorspreferably include the above-described nucleic acid operably linked(under the control of ) a promoter; either constitutive or inducible.The vector can also include an initiation and a termination codon.

[0018] This invention also provides for an isolated human NBCCS (PTC)polypeptide, said polypeptide comprising a subsequence of at least 10contiguous amino acids of a polypeptide encoded by a nucleic acidselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 58, andSEQ. ID NO: 59, or conservative substitutions of said polypeptidesubsequence. This NBCCS polypeptide is preferably at least 50, morepreferably at least 100, and most preferably at least 200, 400, 500, oreven 800 residues (amino acids) in length. The polypeptide can be apolypeptide encoded by a nucleic acid amplified from genomic DNA or anRNA using any of the primers pairs provided in Table 2. In aparticularly preferred embodiment, the NBCCS polypeptide is apolypeptide encoded by a nucleic acid selected from the group consistingof SEQ ID NO: 1, SEQ ID NO: 58, and SEQ. ID NO: 59.

[0019] This invention also includes an isolated NBCCS (PTC) polypeptidecomprising at least 10 contiguous amino acids from a polypeptidesequence encoded by a nucleic acid selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 58, and SEQ. ID NO: 59, wherein: saidpolypeptide, when presented as an antigen, elicits the production of anantibody which specifically binds to a polypeptide encoded by a nucleicacid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 58,and SEQ. ID NO: 59; and said polypeptide does not bind to antiseraraised against a polypeptide encoded by a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 58, and SEQ. IDNO: 59 which has been fully immunosorbed with a polypeptide encoded by asequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:58, and SEQ. ID NO: 59. This polypeptide is preferably at least 50, morepreferably at least 100, and most preferably at least 200, 400, 500, oreven 800 amino acid residues in length. In a particularly preferredembodiment, this polypeptide is encoded by a nucleic acid selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 58, and SEQ. ID NO: 59.

[0020] The polypeptides of this invention can include conservativesubstitutions of any of the above-described polypeptides. In aparticularly preferred embodiment the above-described nucleic acidsand/or proteins, or subsequences thereof, are not a PTC nucleic acid orpolypeptide from a Drosophila, a murine, or C. elegans.

[0021] In another embodiment, this invention provides for anti-NBCCSantibodies. Particularly preferred antibodies specifically bind apolypeptide comprising at least 10, more preferably at least 20, 40, 50,and most preferably at least 100, 200, 400, and even 800 contiguousamino acids, or even the full length polypeptide encoded by a nucleicacid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 58,and SEQ. ID NO: 59 wherein: said polypeptide, when presented as anantigen, elicits the production of an antibody which specifically bindsto a polypeptide encoded by a nucleic acid selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 58, and SEQ. ID NO: 59; and saidpolypeptide does not bind to antisera raised against a polypeptideencoded by a nucleic acid sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 58, and SEQ. ID NO: 59 which has been fullyimmunosorbed with a polypeptide encoded by a nucleic acid sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 58, andSEQ. ID NO: 59. The antibody can be polyclonal or monoclonal. Theantibody can also be humanized or human.

[0022] This invention also provides for cells (e.g., recombinant cellssuch as hybridomas or triomas) expressing any of the above-describedantibodies.

[0023] This invention also provides for methods of detecting apredisposition to nevoid basal cell carcinoma syndrome (NBCCS) or to abasal cell carcinoma. The methods include the steps of i) providing abiological sample of the organism; and ii) detecting a human NBCCS (PTC)gene or gene product in the sample. The provision of a biological sampleand detection methods are described herein. In particular, detecting caninvolve detecting the presence or absence, or quantifying an NBCCS geneor subsequence thereof including any of the above-described nucleicacids. The detecting can also involve detecting the presence or absenceor quantifying a NBCCS polypeptide or subsequence thereof including anyof the above-described polypeptides. The detecting can involve detectingthe presence or absence of normal or abnormal NBCCS nucleic acids orpolypeptides. For example, one can detect a predisposition to BCC orNBCCS by detecting the presence of a mutation in a NBCCS nucleic acid.Particularly preferred assays include hybridization assays and/orsequencing for nucleic acids and immunoassays for NBCCS polypeptides.

[0024] In another embodiment, this invention provides forpharmacological compositions comprising a pharmaceutically acceptablecarrier and a molecule selected from the group consisting of an vectorencoding an NBCCS polypeptide or subsequence thereof, an NBCCSpolypeptide or subsequence thereof, and an anti-NBCCS antibody asdescribed herein.

[0025] This invention also provides for primers for the amplification ofone or more exons of the NBCCS (PTC) gene. These primers include, butare not limited to the primers provided in Table 2.

[0026] This invention also provides kits for the detection and/orquantification of NBCCS gene or gene product. The kits can include acontainer containing one or more of any of the above identified nucleicacids, amplification primers, and antibodies with or without labels,free, or bound to a solid support as described herein. The kits can alsoinclude instructions for the use of one or more of these reagents in anyof the assays described herein.

[0027] Finally, this invention also provides therapeutic methods. Theseinclude a methods of treating basal cell carcinoma and/or nevoid basalcell carcinoma syndrome and/or solar keratoses in a mammal. The methodscan involve transfecting cells of the mammal with a vector expressing anevoid basal cell carcinoma syndrome (NBCCS) polypeptide such that thecells express a functional NBCCS polypeptide as described herein. Thetransfection can be in vivo or ex vivo. Ex vivo transfection ispreferably followed by re-infusion of the cells back into the organismas described herein. Other methods involve administering to the mammal atherapeutically effective dose of a composition comprising a NBCCS (PTC)polypeptide and a pharmacological excipient as described herein. Themethods are preferably performed on mammals such as mice, rats, rabbits,sheep, goats, pigs, more preferably on primates including humanpatients.

Definitions

[0028] The term “antibody” refers to a polypeptide substantially encodedby an immunoglobulin gene or immunoglobulin genes, or fragments thereofwhich specifically bind and recognize an analyte (antigen). Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively.

[0029] An exemplary immunoglobulin (antibody) structural unit comprisesa tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

[0030] Antibodies exist e.g., as intact immunoglobulins or as a numberof well characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂ a dimer of Fabwhich itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially an Fab withpart of the hinge region (see, Fundamental Immunology, Third Edition, W.E. Paul, ed., Raven Press, N.Y. 1993). While various antibody fragmentsare defined in terms of the digestion of an intact antibody, one ofskill will appreciate that such fragments may be synthesized de novoeither chemically or by utilizing recombinant DNA methodology. Thus, theterm antibody, as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies or those synthesized denovo using recombinant DNA methodologies (e.g., single chain Fv).

[0031] An “anti-NBCCs” antibody is an antibody or antibody fragment thatspecifically binds a polypeptide encoded by the NBCCS gene, cDNA, orsubsequence thereof.

[0032] A “chimeric antibody” is an antibody molecule in which (a) theconstant region, or a portion thereof, is altered, replaced or exchangedso that the antigen binding site (variable region) is linked to aconstant region of a different or altered class, effector functionand/or species, or an entirely different molecule which confers newproperties to the chimeric antibody, e.g., an enzyme, toxin, hormone,growth factor, drug, etc.; or (b) the variable region, or a portionthereof, is altered, replaced or exchanged with a variable region havinga different or altered antigen specificity.

[0033] The term “immunoassay” is an assay that utilizes an antibody tospecifically bind an analyte. The immunoassay is characterized by theuse of specific binding properties of a particular antibody to isolate,target, and/or quantify the analyte.

[0034] The terms “isolated” “purified” or “biologically pure” refer tomaterial which is substantially or essentially free from componentswhich normally accompany it as found in its native state.

[0035] The term “nucleic acid” refers to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form, andunless otherwise limited, encompasses known analogs of naturalnucleotides that can function in a similar manner as naturally occurringnucleotides.

[0036] The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

[0037] A “label” is a composition detectable by spectroscopic,photochemical, biochemical, immunochemical, or chemical means. Forexample, useful labels include ³²P, fluorescent dyes, electron-densereagents, enzymes (e.g., as commonly used in an ELISA), biotin,dioxigenin, or haptens and proteins for which antisera or monoclonalantibodies are available (e.g., the peptide of SEQ ID NO 1 can be madedetectable, e.g., by incorporating a radio-label into the peptide, andused to detect antibodies specifically reactive with the peptide).

[0038] As used herein a “nucleic acid probe” is defined as a nucleicacid capable of binding to a target nucleic acid of complementarysequence through one or more types of chemical bonds, usually throughcomplementary base pairing, usually through hydrogen bond formation. Asused herein, a probe may include natural (i.e. A, G, C, or T) ormodified bases (7-deazaguanosine, inosine, etc.). In addition, the basesin a probe may be joined by a linkage other than a phosphodiester bond,so long as it does not interfere with hybridization. Thus, for example,probes may be peptide nucleic acids in which the constituent bases arejoined by peptide bonds rather than phosphodiester linkages. It will beunderstood by one of skill in the art that probes may bind targetsequences lacking complete complementarity with the probe sequencedepending upon the stringency of the hybridization conditions. Theprobes are preferably directly labeled as with isotopes, chromophores,lumiphores, chromogens, or indirectly labeled such as with biotin towhich a streptavidin complex may later bind. By assaying for thepresence or absence of the probe, one can detect the presence or absenceof the select sequence or subsequence.

[0039] A “labeled nucleic acid probe” is a nucleic acid probe that isbound, either covalently, through a linker, or through ionic, van derWaals or hydrogen bonds to a label such that the presence of the probemay be detected by detecting the presence of the label bound to theprobe.

[0040] The term “target nucleic acid” refers to a nucleic acid (oftenderived from a biological sample), to which a nucleic acid probe isdesigned to specifically hybridize. It is either the presence or absenceof the target nucleic acid that is to be detected, or the amount of thetarget nucleic acid that is to be quantified. The target nucleic acidhas a sequence that is complementary to the nucleic acid sequence of thecorresponding probe directed to the target. The term target nucleic acidmay refer to the specific subsequence of a larger nucleic acid to whichthe probe is directed or to the overall sequence (e.g., gene or mRNA)whose expression level it is desired to detect. The difference in usagewill be apparent from context.

[0041] “Subsequence” refers to a sequence of nucleic acids or aminoacids that comprise a part of a longer sequence of nucleic acids oramino acids (e.g., polypeptide) respectively.

[0042] The term “recombinant” when used with reference to a cell, ornucleic acid, or vector, indicates that the cell, or nucleic acid, orvector, has been modified by the introduction of a heterologous nucleicacid or the alteration of a native nucleic acid, or that the cell isderived from a cell so modified. Thus, for example, recombinant cellsexpress genes that are not found within the native (non-recombinant)form of the cell or express native genes that are otherwise abnormallyexpressed, under expressed or not expressed at all.

[0043] The term “identical” in the context of two nucleic acids orpolypeptide sequences refers to the residues in the two sequences whichare the same when aligned for maximum correspondence. Optimal alignmentof sequences for comparison can be conducted, e.g., by the localhomology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482,by the homology alignment algorithm of Needleman and Wunsch (1970) J.Mol. Biol. 48:443, by the search for similarity method of Pearson andLipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by inspection.

[0044] An additional algorithm that is suitable for determining sequencesimilarity is the BLAST algorithm, which is described in Altschul et al.(1990) J. Mol. Biol. 215: 403-410. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence that eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al, supra.). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a word length (W) of11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands.

[0045] The BLAST algorithm performs a statistical analysis of thesimilarity between two sequences; see, e.g., Karlin and Altschul (1993)Proc. Nat'l. Acad. Sci. USA 90: 5873-5787. One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to an NBCCS gene or cDNAif the smallest sum probability in a comparison of the test nucleic acidto an NBCCS nucleic acid (e.g., SEQ ID Nos: 1, 58, or 59) is less thanabout 1, preferably less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

[0046] The term “substantial identity” or “substantial similarity” inthe context of a polypeptide indicates that a polypeptides comprises asequence with at least 70% sequence identity to a reference sequence, orpreferably 80%, or more preferably 85% sequence identity to thereference sequence, or most preferably 90% identity over a comparisonwindow of about 10-20 amino acid residues. An indication that twopolypeptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a polypeptide is substantially identical to a secondpolypeptide, for example, where the two peptides differ only by aconservative substitution.

[0047] An indication that two nucleic acid sequences are substantiallyidentical is that the polypeptide which the first nucleic acid encodesis immunologically cross reactive with the polypeptide encoded by thesecond nucleic acid.

[0048] Another indication that two nucleic acid sequences aresubstantially identical is that the two molecules hybridize to eachother under stringent conditions.

[0049] “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetpolynucleotide sequence.

[0050] The phrase “hybridizing specifically to”, refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex mixture (e.g., total cellular) DNA or RNA. The term “stringentconditions” refers to conditions under which a probe will hybridize toits target subsequence, but to no other sequences. Stringent conditionsare sequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. The Tm is the temperature (under definedionic strength, pH, and nucleic acid concentration) at which 50% of theprobes complementary to the target sequence hybridize to the targetsequence at equilibrium. (As the target sequences are generally presentin excess, at Tm, 50% of the probes are occupied at equilibrium).Typically, stringent conditions will be those in which the saltconcentration is less than about 1.0 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide.

[0051] The phrases “specifically binds to a protein” or “specificallyimmunoreactive with”, when referring to an antibody refers to a bindingreaction which is determinative of the presence of the protein in thepresence of a heterogeneous population of proteins and other biologics.Thus, under designated immunoassay conditions, the specified antibodiesbind preferentially to a particular protein and do not bind in asignificant amount to other proteins present in the sample. Specificbinding to a protein under such conditions requires an antibody that isselected for its specificity for a particular protein. A variety ofimmunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select monoclonal antibodiesspecifically immunoreactive with a protein. See Harlow and Lane (1988)Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork, for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity.

[0052] A “conservative substitution”, when describing a protein refersto a change in the amino acid composition of the protein that does notsubstantially alter the protein's activity. Thus, “conservativelymodified variations” of a particular amino acid sequence refers to aminoacid substitutions of those amino acids that are not critical forprotein activity or substitution of amino acids with other amino acidshaving similar properties (e.g., acidic, basic, positively or negativelycharged, polar or non-polar, etc.) such that the substitutions of evencritical amino acids do not substantially alter activity. Conservativesubstitution tables providing functionally similar amino acids are wellknown in the art. The following six groups each contain amino acids thatare conservative substitutions for one another:

[0053] 1) Alanine (A), Serine (S), Threonine (T);

[0054] 2) Aspartic acid (D), Glutamic acid (E);

[0055] 3) Asparagine (N), Glutamine (Q);

[0056] 4) Arginine (R), Lysine (K);

[0057] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0058] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0059] See also, Creighton (1984) Proteins, W. H. Freeman and Company.In addition, individual substitutions, deletions or additions whichalter, add or delete a single amino acid or a small percentage of aminoacids in an encoded sequence are also “conservatively modifiedvariations”.

[0060] The terms human “PTC” or human “NBCCS gene or cDNA” are usedinterchangeably to refer to the human homologue of the Drosophilapatched (ptc) gene disclosed herein. As explained below, the human PTCgene is a tumor suppressor gene also involved in the etiology of nevoidbasal carcinoma cell syndrome.

[0061] A “gene product”, as used herein, refers to a nucleic acid whosepresence, absence, quantity, or nucleic acid sequence is indicative of apresence, absence, quantity, or nucleic acid composition of the gene.Gene products thus include, but are not limited to, an mRNA transcript,a cDNA reverse transcribed from an mRNA, an RNA transcribed from thatcDNA, a DNA amplified from the cDNA, an RNA transcribed from theamplified DNA or subsequences of any of these nucleic acids.Polypeptides expressed by the gene or subsequences thereof are also geneproducts. The particular type of gene product will be evident from thecontext of the usage of the term.

[0062] An “abnormal PTC (or NBCCS) gene or cDNA” refers to a NBCCS geneor cDNA that encodes a non-functional NBCCS polypeptide, or an NBCCSpolypeptide of substantially reduced functionality. Non-functional, orreduced functionality, NBCCS polypeptides are characterized by apredisposition (i.e., an increased likelihood as compared to the“normal” population) for, or the onset of, nevoid basal cell carcinomasyndrome. Similarly, “abnormal PTC (or NBCCS) gene product” refers to anucleic acid encoding a non-functional or reduced functionality NBCCSpolypeptide or the non-functional or reduced functionality NBCCSpolypeptide itself. Abnormal NBCCS (PTC) genes or gene products include,for example, NBCCS genes or subsequences altered by mutations (e.g.insertions, deletions, point mutations, etc.), splicing errors,premature termination codons, missing initiators, etc. Abnormal NBCCSpolypeptides include polypeptides expressed by abnormal NBCCS genes ornucleic acid gene products or subsequences thereof. Abnormal expressionof NBCCS genes includes underexpression (as compared to the “normal”healthy population) of NBCCS e.g., through partial or completeinactivation, haploinsufficiency, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

[0063]FIG. 1 shows an integrated framework map of the NBCCS region. Bothlinkage and tumor deletion studies place NBCCS between D9SJ96 and D9S180but are conflicting, with regard to whether the gene lies proximal ordistal to D9S287. The order of six polymorphic markers, D9S197,D9S196-D9S280-FACC-D9S287-D9S180, is derived from genetic linkage data(Farndon et al., 1994; Pericak-Vance et al., 1995). D9S196 and D9S197show no measurable recombination. Pulsed-field gel electrophoresis(PFGE) and FISH give a minimum distance of 2 Mb between D9S196 andD9S180. Key information about YAC, BAC, and cosmid contigs in the NBCCSregion is shown. In total, 22 overlapping YACs and more than 800 cosmidswere isolated from this region. BAC and cosmid contigs covering morethan 1.2 Mb have been submitted to the Genome Data Base.

[0064]FIG. 2 shows a map of the PTC locus. The gene lies on 4overlapping cosmids including 226G7, 42H1 1, 55A16, and 96F9 (LL09NCO1,96-well coordinates). The coding exons of the gene are shown as filledboxes and non-coding (untranslated) exons as open boxes. Splice variantsof the 5′ non-coding, region of the gene indicate at least two alternatefirst exons and possibly a third alternate exon (see Example 2).

[0065]FIG. 3 provides a map of the promoter region of the NBCCS (PTC)gene.

[0066]FIG. 4 illustrates segregation of a premature termination mutationof PTC in an NBCCS pedigree. The C1081T (Q210X) mutation segregating inthis kindred creates a Bfal site. PCR with flanking primers produces a260 bp product, which digests to 171 bp and 90 bp fragments in affectedfamily members. The PCR product remains undigested in unaffected familymembers.

[0067]FIG. 5 shows a frameshift mutation of PTC in a sporadic NBCCSpatient. (FIG. 5A) Both parents (1 and 2) of an affected individual (3)were free of phenotypic features of NBCCS. (FIG. 5B) The patient washeterozygous for a single stranded conformation polymorphism (SSCP)variant in Exon 12 that was not present in her parents, and sequencingof a PCR product from genomic DNA showed two sequences out of framefollowing base 2000. The abnormal conformer was sequenced and containeda 1 bp insertion resulting in a premature stop nine amino acidsdownstream. Sequences of PCR products from both parents were normal.

[0068]FIG. 6 shows an ultraviolet B-induced mutation of PTC in asporadic basal cell carcinoma. (FIG. 6A) CC to TT mutation in theremaining allele with allelic loss of the NBCCS region. This DNAalteration, which results in a premature stop, is typical of ultravioletB mutagenesis. (FIG. 6B) Constitutional DNA from the patient has anormal sequence.

[0069]FIG. 7 shows a PTC deletion in a sporadic BCC. (FIG. 7A) A 14 bpdeletion in the remaining allele of a tumor with allelic loss of theNBCCS region. Despite the fact that this tumor was removed from thenose, a highly sunlight-exposed area, the mutation cannot be relatedspecifically to ultraviolet radiation. (FIG. 7B) Constitutional DNA hadthe normal sequence.

[0070]FIG. 8 shows the nucleotide sequence (SEQ ID NO: 1) of the humanPTC cDNA (Genbank Accession No. U43148). The sequence of PTC is shown,including the open reading frame and flanking 5′ and 3′ sequences. Thecorresponding amino acid sequence is presented in SEQ ID NO: 60.

[0071]FIG. 9 shows mutations in the human PTC gene in DNA obtained fromdesmoplastic medulloblastomas D322, D292 and D86. FIG. 9A:microsatellite D9S302 shows loss of heterozygosity in tumor D322. Inthis tumor, the remaining allele of exon 6 exhibits an altered mobility.DNA sequencing shows a single base pair deletion in the tumor DNA whichresults in a frameshift and resulting truncation of the PTC protein.FIG. 9B shows SSCP variants in exon 10 in tumor D292 without allelicloss of chromosome 9q. sequencing of the altered allele shows a fourbase pair insertion at position 1393. FIG. 9C shows the sequencing ofexon 10 in tumor D86. This tumor, which has LOH, has a six base pairin-frame deletion at position 1444 (CTG GGC). This leads to a deletionof two amino acids (glycine and leucine) in transmembrane region 3.

[0072]FIG. 10 shows PTC mRNA levels as determined using asemi-quantitative RT-PCR approach as described in Example 5. 250 ng ofRNA in 10 μl was reverse transcribed in the presence of 40 pg of each ofthe standard RNAs with internal deletions for PTC, β₂-microglobin andGAPDH. The cDNAs were then amplified with primers for PTC and thehousekeeping genes. The products were separated and quantitated on anABI 373A sequencer. The expression level of the three genes weredetermined as the ratio of signals of the sample (right peaks) to thespecific standards (left peaks). FIG. 10A: PTC mRNA expression in tworepresentative tumors of the classical variant of MB; FIG. 10B:expression in desmoplastic MBs; FIG. 10C: expression in adult humancerebellum.

[0073]FIG. 11 is a schematic diagram of the PTC protein with thelocation of germ line mutations indicated. The putative transmembranedomains are designated “TM1” to “TM12”; the hatched boxes represent thetwo putative extracellular loops. An asterisk (*) denotes a missense; atriangle (▾) denotes a nonsense or frameshift; and a diamond (♦) denotesputative splicing variants. Open boxes above correspond to exonsencoding relevant domains.

DETAILED DESCRIPTION

[0074] This invention pertains to the discovery of a tumor suppressorgene associated with the etiology of nevoid basal cell carcinomasyndrome. In addition, this invention pertains to the discovery thatvarious cancers, including sporadic basal cell carcinomas (BCCs) canarise with somatic loss of both copies of the same gene.

[0075] Nevoid basal cell carcinoma syndrome, also known as Gorlinsyndrome and the basal cell nevus syndrome, is an autosomal dominantdisorder that predisposes to both cancer and developmental defects(Gorlin (1995) Dermatologic Clinics, 13: 113-125). Its prevalence hasbeen estimated at 1 per 56,000, and 1-2% of medulloblastomas and 0.5% ofbasal cell carcinomas (BCCS) are attributable to the syndrome (Springate(1986) J. Pediatr. Surg. 21: 908-910; Evans et al. (1991) British J.Cancer., 64: 959-961). In addition to basal cell carcinomas (BCCs) andmedulloblastomas, NBCCS patients are also at an increased risk forovarian fibromas, meningiomas, fibrosarcomas, rhabdomyosarcomas, cardiacfibromas and ovarian dermoids (Evans et al. (1991) supra., Evans et al.(1993) J. Med. Genet. 30: 460-464; Gorlin (1995) supra.).

[0076] Implications for the affected individual can be severe,predominantly due to the prolific basal cell carcinomas which can numbermore than 500 in a lifetime (Shanley et al. (1994) supra). Expression ofmany features of the syndrome is variable, but the severity tends tobreed true within families (Anderson et al. (1967) Am. J. Hum. Genet.,19: 12-22). This variation between families may reflect specificphenotypic effects of different mutations, modifier genes, orenvironmental factors (sunlight exposure is likely to play a role in theage of onset and incidence of basal cell carcinomas). One third to onehalf of patients have no affected relatives and are presumed to be theproduct of new germ cell mutations (Gorlin (1995) supra.). Unilateraland segmental NBCCS are attributed to somatic mutation in one cell of anearly embryo (Gutierrez and Mora, (1986) supra.)

I. Uses of the NBCCS (PTC) cDNA

[0077] As indicated above, the NBCCS gene of this invention is a tumorsuppressor gene. Defects in the expression of this gene are associatedthe onset of various cancers, particularly with sporadic basal cellcarcinoma in somatic cells. Heritable defects in the expression of theNBCCS gene are a causal factor in the etiology of NBCCS and itsattendant developmental abnormalities (see example 2, below).

[0078] While basal cell carcinomas and many features of NBCCS arebelieved to be due to homozygous inactivation of PTC generalized orsymmetric features such as overgrowth, macrocephaly, and facialdysmorphology are believed to be due to haploinsufficiency or othermechanisms of partial gene inactivation.

[0079] Clearly, detection of defective NBCCS (PTC) gene expression is ofclinical value. The presence of an NBCCS (PTC) gene, cDNA, mRNA,protein, or subsequence of the gene, cDNA, or protein in a biologicalsample is useful, e.g., as a marker to asses in vivo and/or in situ RNAtranscription and/or translation, in cancer diagnostics (as in thedetection or verification of basal cell carcinoma), in prophpylaxis forNBCCS or BCC as an indication of a heritable predilection for NBCCS orBCC, or in DNA forensic analysis such as DNA fingerprinting. Full-lengthNBCCS cDNA, individual exons, or subsequences thereof are also useful asprobes (particularly when labeled) for the detection of the presence orabsence and/or quantitation of normal or abnormal (e.g., truncated ormutated) NBCCS (PTC) DNA or RNA in a biological sample. The labeledprobes can also be useful as in fluorescent karyotyping analysis asmarkers of the NBCCS gene. Because the NBCCS cDNA or subsequencesthereof is shown herein to map to human chromosome 9q22.3, one of skillcan use the gene, cDNA, or subsequences, as a probe to asses whetherthere are any gross chromosomal abnormalities in this region ofchromosome 9. This is useful, for instance, in in utero screening of afetus to monitor for the presence of chromosomal abnormalities inparticular for a predilection of NBCCS or basal cell carcinomas.

[0080] Similarly, the proteins encoded by the NBCCS cDNA can be used asdiagnostic markers for NBCCS and/or basal cell carcinomas. The proteinsor subsequences thereof can also be used as antigens for raisinganti-NBCCS protein antibodies. The antibodies are useful forimmunoassays for the detection of normal or abnormal expression of NBCCSproteins, and for the isolation of NBCCS polypeptides (as with affinitychromatography).

[0081] Vectors encoding the NBCCS proteins are useful for expressingthose proteins to provide immunogens for antibody production. Vectorsencoding the NBCCS proteins are also useful for transforming cells invitro or in vivo to express NBCCS proteins. In vivo transformation ofcells to express heterologous NBCCS genes can be used to offsetdeficient expression of the NBCCS protein.

[0082] Cells and/or tissues expressing the NBCCS (PTC) gene may be usedto monitor expression levels of NBCCS polypeptides in a wide variety ofcontexts. For example, where the effects of a drug on NBCCS expressionis to be determined the drug will be administered to the transformed (toexpress NBCCS) organism, tissue, or cell. Expression levels, orexpression products will be assayed as described below and the resultscompared results from to organisms, tissues, or cells similarly treated,but without the drug being tested.

II. The NBCCS (PTC) Gene

[0083] A) The Human PTC Gene.

[0084] Sequence Listing ID NO: 1 provides both nucleic acid andpolypeptide sequence listings for the human PTC cDNA of this invention.The sequence of human PTC, as shown, consists of an open reading frameof 3888 nucleotides flanked by 441 and 2240 nucleotides on the 5′ endand on the 3′ end, respectively (SEQ ID NO: 1, FIG. 8). The open readingframe of human PTC cDNA encodes for a putative protein of 1296 aminoacids. The open reading frame starts with an ATG codon that has amoderate match for the translational start consensus sequence invertebrates (GAGGCTATGT (SEQ ID NO: 6) in PTC versus GCCGCCATGG (SEQ IDNO: 7) (Kozak (1991) J. Biol. Chem. 266:19867-19870)). This codon codesfor the first amino acid of one human form of the PTC protein consistingof 1296 amino acids with a relative molecular weight (M_(r)) of 131×10³.It shows 61% sequence identity to its Drosophila counterpart. The openreading frame extends an additional 354 nucleotides upstream of the ATGcodon (starting at base pair 88 of the sequence shown in FIG. 8). The 3′untranslated region contains a canonical polyadenylation signal (AATAAA(SEQ ID NO: 8)) as well as mRNA destabilizing (ATTTA (SEQ ID NO: 9))motifs. These are localized 1401 nucleotides and 547, 743, and 1515nucleotides after the termination codon, respectively. A second humanPTC protein contains an open reading frame that continues right throughto the 5′ end, and may be initiated by upstream sequences.

[0085] B) Isolation of cDNA and/or Probes.

[0086] The nucleic acids (e.g., NBCCS cDNA, or subsequences (probes)) ofthe present invention are cloned, or amplified by in vitro methods, suchas the polymerase chain reaction (PCR), the ligase chain reaction (LCR),the transcription-based amplification system (TAS), the self-sustainedsequence replication system (SSR). A wide variety of cloning and invitro amplification methodologies are well-known to persons of skill.Examples of these techniques and instructions sufficient to directpersons of skill through many cloning exercises are found in Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology 152Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989)Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook et al.);Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel); Cashionet al., U.S. Pat. No. 5,017,478; and Carr, European Patent No.0,246,864. Examples of techniques sufficient to direct persons of skillthrough in vitro amplification methods are found in Berger, Sambrook,and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202;PCR Protocols A Guide to Methods and Applications (Innis et al. eds)Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson(Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3: 81-94;(Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al.(1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J.Clin. Chem., 35: 1826; Landegren et al., (1988) Science, 241: 1077-1080;Van Brunt (1990) Biotechnology, 8: 291-294; Wu and Wallace, (1989) Gene,4: 560; and Barringer et al. (1990) Gene, 89: 117.

[0087] In one preferred embodiment, the human NBCCS (PTC) cDNA can beisolated by routine cloning methods. The cDNA sequence provided in SEQID NO: 1 can be used to provide probes that specifically hybridize tothe NBCCS gene, in a genomic DNA sample, or to the NBCCS mRNA, in atotal RNA sample (e.g., in a Southern blot). Once the target NBCCSnucleic acid is identified (e.g., in a Southern blot), it can beisolated according to standard methods known to those of skill in theart (see, e.g., Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual, 2nd Ed., Vols. 1-3, Cold Spring Harbor Laboratory; Berger andKimmel (1987) Methods in Enzymology, Vol. 152: Guide to MolecularCloning Techniques, San Diego: Academic Press, Inc.; or Ausubel et al.(1987) Current Protocols in Molecular Biology, Greene Publishing andWiley-Interscience, New York). Methods of screening human cDNA librariesfor the NBCCS gene are provided in Example 1.

[0088] In another preferred embodiment, the human PTC cDNA can beisolated by amplification methods such as polymerase chain reaction(PCR). Table 2 provides primers suitable for the amplification of all 21exons of the cDNA. In addition, appropriate PCR protocols are providedin Example 2.

[0089] C) Labeling of Nucleic Acid Probes.

[0090] Where the NBCCS cDNA or its subsequences are to be used asnucleic acid probes, it is often desirable to label the sequences withdetectable labels. The labels may be incorporated by any of a number ofmeans well known to those of skill in the art. However, in a preferredembodiment, the label is simultaneously incorporated during theamplification step in the preparation of the sample nucleic acids. Thus,for example, polymerase chain reaction (PCR) with labeled primers orlabeled nucleotides will provide a labeled amplification product. Inanother preferred embodiment, transcription amplification using alabeled nucleotide (e.g. fluorescein-labeled UTP and/or CTP)incorporates a label into the transcribed nucleic acids.

[0091] Alternatively, a label may be added directly to an originalnucleic acid sample (e.g., mRNA, polyA mRNA, cDNA, etc.) or to theamplification product after the amplification is completed. Means ofattaching labels to nucleic acids are well known to those of skill inthe art and include, for example nick translation or end-labeling (e.g.with a labeled RNA) by kinasing of the nucleic acid and subsequentattachment (ligation) of a nucleic acid linker joining the samplenucleic acid to a label (e.g., a fluorophore).

[0092] Detectable labels suitable for use in the present inventioninclude any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Useful labels in the present invention include biotin for staining withlabeled streptavidin conjugate, magnetic beads (e.g., Dynabeads™),fluorescent dyes (e.g., fluorescein, texas red, rhodamine, greenfluorescent protein, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S,¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase, alkalinephosphatase and others commonly used in an ELISA), and colorimetriclabels such as colloidal gold or colored glass or plastic (e.g.,polystyrene, polypropylene, latex, etc.) beads. Patents teaching the useof such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241.

[0093] Means of detecting such labels are well known to those of skillin the art. Thus, for example, radiolabels may be detected usingphotographic film or scintillation counters, fluorescent markers may bedetected using a photodetector to detect emitted light. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

III. Antibodies to the NBCCS Polypeptide(s)

[0094] Antibodies are raised to the NBCCS polypeptides of the presentinvention, including individual, allelic, strain, or species variants,and fragments thereof, both in their naturally occurring (full-length)forms and in recombinant forms. Additionally, antibodies are raised tothese polypeptides in either their native configurations or innon-native configurations. Anti-idiotypic antibodies can also begenerated. Many methods of making antibodies are known to persons ofskill. The following discussion is presented as a general overview ofthe techniques available; however, one of skill will recognize that manyvariations upon the following methods are known.

[0095] A) Antibody Production

[0096] A number of immunogens are used to produce antibodiesspecifically reactive with NBCCS polypeptides. Recombinant or syntheticpolypeptides of 10 amino acids in length, or greater, selected fromamino acid sub-sequences of SEQ ID NO 1 are the preferred polypeptideimmunogen (antigen) for the production of monoclonal or polyclonalantibodies. In one class of preferred embodiments, an immunogenicpeptide conjugate is also included as an immunogen. Naturally occurringpolypeptides are also used either in pure or impure form.

[0097] Recombinant polypeptides are expressed in eukaryotic orprokaryotic cells (as described below) and purified using standardtechniques. The polypeptide, or a synthetic version thereof, is theninjected into an animal capable of producing antibodies. Eithermonoclonal or polyclonal antibodies can be generated for subsequent usein immunoassays to measure the presence and quantity of the polypeptide.

[0098] Methods of producing polyclonal antibodies are known to those ofskill in the art. In brief, an immunogen (antigen), preferably apurified polypeptide, a polypeptide coupled to an appropriate carrier(e.g., GST, keyhole limpet hemanocyanin, etc.), or a polypeptideincorporated into an immunization vector such as a recombinant vacciniavirus (see, U.S. Pat. No. 4,722,848) is mixed with an adjuvant andanimals are immunized with the mixture. The animal's immune response tothe immunogen preparation is monitored by taking test bleeds anddetermining the titer of reactivity to the polypeptide of interest. Whenappropriately high titers of antibody to the immunogen are obtained,blood is collected from the animal and antisera are prepared. Furtherfractionation of the antisera to enrich for antibodies reactive to thepolypeptide is performed where desired (see, e.g., Coligan (1991)Current Protocols in Immunology Wiley/Greene, NY; and Harlow and Lane(1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY).

[0099] Antibodies, including binding fragments and single chainrecombinant versions thereof, against predetermined fragments of NBCCSpolypeptides are raised by immunizing animals, e.g., with conjugates ofthe fragments with carrier proteins as described above. Typically, theimmunogen of interest is a peptide of at least about 5 amino acids, moretypically the peptide is 10 amino acids in length, preferably, thefragment is 15 amino acids in length and more preferably the fragment is20 amino acids in length or greater. The peptides are typically coupledto a carrier protein (e.g., as a fusion protein), or are recombinantlyexpressed in an immunization vector. Antigenic determinants on peptidesto which antibodies bind are typically 3 to 10 amino acids in length.

[0100] Monoclonal antibodies are prepared from cells secreting thedesired antibody. These antibodies are screened for binding to normal ormodified polypeptides, or screened for agonistic or antagonisticactivity, e.g., activity mediated through a NBCCS protein. Specificmonoclonal and polyclonal antibodies will usually bind with a K_(D) ofat least about 0.1 mM, more usually at least about 50 μM, and mostpreferably at least about 1 μM or better.

[0101] In some instances, it is desirable to prepare monoclonalantibodies from various mammalian hosts, such as mice, rodents,primates, humans, etc. Description of techniques for preparing suchmonoclonal antibodies are found in, e.g., Stites et al. (eds.) Basic andClinical Immunology (4th ed.) Lange Medical Publications, Los Altos,Calif., and references cited therein; Harlow and Lane, supra; Goding(1986) Monoclonal Antibodies: Principles and Practice (2d ed.) AcademicPress, New York, N.Y.; and Kohler and Milstein (1975) Nature 256:495-497. Summarized briefly, this method proceeds by injecting an animalwith an immunogen. The animal is then sacrificed and cells taken fromits spleen, which are fused with myeloma cells. The result is a hybridcell or “hybridoma” that is capable of reproducing in vitro. Thepopulation of hybridomas is then screened to isolate individual clones,each of which secrete a single antibody species to the immunogen. Inthis manner, the individual antibody species obtained are the productsof immortalized and cloned single B cells from the immune animalgenerated in response to a specific site recognized on the immunogenicsubstance.

[0102] Alternative methods of immortalization include transformationwith Epstein Barr Virus, oncogenes, or retroviruses, or other methodsknown in the art. Colonies arising from single immortalized cells arescreened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells is enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate (preferablymammalian) host. The polypeptides and antibodies of the presentinvention are used with or without modification, and include chimericantibodies such as humanized murine antibodies.

[0103] Other suitable techniques involve selection of libraries ofrecombinant antibodies in phage or similar vectors (see, e.g., Huse etal. (1989) Science 246: 1275-1281; and Ward, et al. (1989) Nature 341:544-546; and Vaughan et al. (1996) Nature Biotechnology, 14: 309-314).

[0104] Frequently, the polypeptides and antibodies will be labeled byjoining, either covalently or non-covalently, a substance which providesfor a detectable signal. A wide variety of labels and conjugationtechniques are known and are reported extensively in both the scientificand patent literature. Suitable labels include radionucleotides,enzymes, substrates, cofactors, inhibitors, fluorescent moieties,chemiluminescent moieties, magnetic particles, and the like. Patentsteaching the use of such labels include U.S. Pat. Nos. 3,817,837;3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.Also, recombinant immunoglobulins may be produced. See, Cabilly, U.S.Pat. No. 4,816,567; and Queen etal. (1989) Proc. Nat'l Acad. Sci. USA86: 10029-10033.

[0105] The antibodies of this invention are also used for affinitychromatography in isolating NBCCS polypeptides. Columns are prepared,e.g., with the antibodies linked to a solid support, e.g., particles,such as agarose, Sephadex, or the like, where a cell lysate is passedthrough the column, washed, and treated with increasing concentrationsof a mild denaturant, whereby purified NBCCS polypeptides are released.

[0106] The antibodies can be used to screen expression libraries forparticular expression products such as normal or abnormal human NBCCSprotein. Usually the antibodies in such a procedure are labeled with amoiety allowing easy detection of presence of antigen by antibodybinding.

[0107] Antibodies raised against NBCCS polypeptides can also be used toraise anti-idiotypic antibodies. These are useful for detecting ordiagnosing various pathological conditions related to the presence ofthe respective antigens.

[0108] B) Human or Humanized (chimeric) Antibody Production.

[0109] The anti-NBCCS antibodies of this invention can also beadministered to an organism (e.g., a human patient) for therapeuticpurposes (e.g., to block the action an NBCCS polypeptide or as targetingmolecules when conjugated or fused to effector molecules such as labels,cytotoxins, enzymes, growth factors, drugs, etc.). Antibodiesadministered to an organism other than the species in which they areraised are often immunogenic. Thus, for example, murine antibodiesadministered to a human often induce an immunologic response against theantibody (e.g., the human anti-mouse antibody (HAMA) response) onmultiple administrations. The immunogenic properties of the antibody arereduced by altering portions, or all, of the antibody intocharacteristically human sequences thereby producing chimeric or humanantibodies, respectively.

[0110] i) Humanized (chimeric) Antibodies.

[0111] Humanized (chimeric) antibodies are immunoglobulin moleculescomprising a human and non-human portion. More specifically, the antigencombining region (or variable region) of a humanized chimeric antibodyis derived from a non-human source (e.g., murine) and the constantregion of the chimeric antibody (which confers biological effectorfunction to the immunoglobulin) is derived from a human source. Thehumanized chimeric antibody should have the antigen binding (e.g.,anti-NBCCS polypeptide) specificity of the non-human antibody moleculeand the effector function conferred by the human antibody molecule. Alarge number of methods of generating chimeric antibodies are well knownto those of skill in the art (see, e.g., U.S. Pat. Nos: 5,502,167,5,500,362, 5,491,088, 5,482,856, 5,472,693, 5,354,847, 5,292,867,5,231,026, 5,204,244, 5,202,238, 5,169,939, 5,081,235, 5,075,431, and4,975,369).

[0112] In general, the procedures used to produce these chimericantibodies consist of the following steps (the order of some steps maybe interchanged): (a) identifying and cloning the correct gene segmentencoding the antigen binding portion of the antibody molecule; this genesegment (known as the VDJ, variable, diversity and joining regions forheavy chains or VJ, variable, joining regions for light chains (orsimply as the V or Variable region) may be in either the cDNA or genomicform; (b) cloning the gene segments encoding the constant region ordesired part thereof; (c) ligating the variable region to the constantregion so that the complete chimeric antibody is encoded in atranscribable and translatable form; (d) ligating this construct into avector containing a selectable marker and gene control regions such aspromoters, enhancers and poly(A) addition signals; (e) amplifying thisconstruct in a host cell (e.g., bacteria); (f) introducing the DNA intoeukaryotic cells (transfection) most often mammalian lymphocytes; andculturing the host cell under conditions suitable for expression of thechimeric antibody.

[0113] Antibodies of several distinct antigen binding specificities havebeen manipulated by these protocols to produce chimeric proteins (e.g.,anti-TNP: Boulianne et al. (1984) Nature, 312: 643; and anti-tumorantigens: Sahagan et al. (1986) J. Immunol., 137: 1066). Likewiseseveral different effector functions have been achieved by linking newsequences to those encoding the antigen binding region. Some of theseinclude enzymes (Neuberger et al. (1984) Nature 312: 604),immunoglobulin constant regions from another species and constantregions of another immunoglobulin chain (Sharon et al. (1984) Nature309: 364; Tan et al., (1985) J. Immuno. 135: 3565-3567).

[0114] In one preferred embodiment, a recombinant DNA vector is used totransfect a cell line that produces an anti-NBCCS antibody. The novelrecombinant DNA vector contains a “replacement gene” to replace all or aportion of the gene encoding the immunoglobulin constant region in thecell line (e.g., a replacement gene may encode all or a portion of aconstant region of a human immunoglobulin, a specific immunoglobulinclass, or an enzyme, a toxin, a biologically active peptide, a growthfactor, inhibitor, or a linker peptide to facilitate conjugation to adrug, toxin, or other molecule, etc.), and a “target sequence” whichallows for targeted homologous recombination with immunoglobulinsequences within the antibody producing cell.

[0115] In another embodiment, a recombinant DNA vector is used totransfect a cell line that produces an antibody having a desiredeffector function, (e.g., a constant region of a human immunoglobulin)in which case, the replacement gene contained in the recombinant vectormay encode all or a portion of a region of an anti-NBCCS antibody andthe target sequence contained in the recombinant vector allows forhomologous recombination and targeted gene modification within theantibody producing cell. In either embodiment, when only a portion ofthe variable or constant region is replaced, the resulting chimericantibody may define the same antigen and/or have the same effectorfunction yet be altered or improved so that the chimeric antibody maydemonstrate a greater antigen specificity, greater affinity bindingconstant, increased effector function, or increased secretion andproduction by the transfected antibody producing cell line, etc.

[0116] Regardless of the embodiment practiced, the processes ofselection for integrated DNA (via a selectable marker), screening forchimeric antibody production, and cell cloning, can be used to obtain aclone of cells producing the chimeric antibody.

[0117] Thus, a piece of DNA which encodes a modification for amonoclonal antibody can be targeted directly to the site of theexpressed immunoglobulin gene within a B-cell or hybridoma cell line.DNA constructs for any particular modification may be used to alter theprotein product of any monoclonal cell line or hybridoma. Such aprocedure circumvents the costly and time consuming task of cloning bothheavy and light chain variable region genes from each B-cell cloneexpressing a useful antigen specificity. In addition to circumventingthe process of cloning variable region genes, the level of expression ofchimeric antibody should be higher when the gene is at its naturalchromosomal location rather than at a random position. Detailed methodsfor preparation of chimeric (humanized) antibodies can be found in U.S.Pat. No. 5,482,856.

[0118] ii) Human Antibodies.

[0119] In another embodiment, this invention provides for fully humananti-NBCCS antibodies. Human antibodies consist entirely ofcharacteristically human polypeptide sequences. The human anti-NBCCSantibodies of this invention can be produced in using a wide variety ofmethods (see, e.g., Larrick et al., U.S. Pat. No. 5,001,065, forreview).

[0120] In one preferred embodiment, the human anti-NBCCS antibodies ofthe present invention are usually produced initially in trioma cells.Genes encoding the antibodies are then cloned and expressed in othercells, particularly, nonhuman mammalian cells.

[0121] The general approach for producing human antibodies by triomatechnology has been described by Ostberg et al. (1983) Hybridoma 2:361-367, Ostberg, U.S. Pat. No. 4,634,664, and Engelman et al., U.S.Pat. No. 4,634,666. The antibody-producing cell lines obtained by thismethod are called triomas because they are descended from three cells;two human and one mouse. Triomas have been found to produce antibodymore stably than ordinary hybridomas made from human cells.

[0122] Preparation of trioma cells requires an initial fusion of a mousemyeloma cell line with unimmunized human peripheral B lymphocytes. Thisfusion generates a xenogenic hybrid cell containing both human and mousechromosomes (see, Engelman, supra.). Xenogenic cells that have lost thecapacity to secrete antibodies are selected. Preferably, a xenogeniccell is selected that is resistant to 8-azaguanine. Cells possessingresistance to 8-azaguanine are unable to propagate onhypoxanthine-aminopterin-thymidine (HAT) or azaserine-hypoxanthine (AH)media.

[0123] The capacity to secrete antibodies is conferred by a furtherfusion between the xenogenic cell and B-lymphocytes immunized against anNBCCS polypeptide or an epitope thereof. The B-lymphocytes are obtainedfrom the spleen, blood or lymph nodes of human donor. If antibodiesagainst a specific antigen or epitope are desired, it is preferable touse that antigen or epitope thereof as the immunogen rather than NBCCSpolypeptide. Alternatively, B-lymphocytes are obtained from anunimmunized individual and stimulated with an NBCCS polypeptide, or aepitope thereof, in vitro. In a further variation, B-lymphocytes areobtained from an infected, or otherwise immunized individual, and thenhyperimmunized by exposure to an NBCCS polypeptide for about seven tofourteen days, in vitro.

[0124] The immunized B-lymphocytes prepared by one of the aboveprocedures are fused with a xenogenic hybrid cell by well known methods.For example, the cells are treated with 40-50% polyethylene glycol of MW1000-4000, at about 37° C. for about 5-10 min. Cells are separated fromthe fusion mixture and propagated in media selective for the desiredhybrids. When the xenogenic hybrid cell is resistant to 8-azaguanine,immortalized trioma cells are conveniently selected by successivepassage of cells -on HAT or AH medium. Other selective procedures are,of course, possible depending on the nature of the cells used in fusion.Clones secreting antibodies having the required binding specificity areidentified by assaying the trioma culture medium for the ability to bindto an NBCCS polypeptide or an epitope thereof. Triomas producing humanantibodies having the desired specificity are subdloned by the limitingdilution technique and grown in vitro in culture medium, or are injectedinto selected host animals and grown in vivo.

[0125] The trioma cell lines obtained are then tested for the ability tobind an NBCCS polypeptide or an epitope thereof. Antibodies areseparated from the resulting culture medium or body fluids byconventional antibody-fractionation procedures, such as ammonium sulfateprecipitation, DEAE cellulose chromatography and affinitychromatography.

[0126] Although triomas are genetically stable they do not produceantibodies at very high levels. Expression levels can be increased bycloning antibody genes from the trioma into one or more expressionvectors, and transforming the vector into a cell line such as the celllines typically used for expression of recombinant or humanizedimmunoglobulins. As well as increasing yield of antibody, this strategyoffers the additional advantage that immunoglobulins are obtained from acell line that does not have a human component, and does not thereforeneed to be subjected to the especially extensive viral screeningrequired for human cell lines.

[0127] The genes encoding the heavy and light chains of immunoglobulinssecreted by trioma cell lines are cloned according to methods, includingthe polymerase chain reaction, known in the art (see, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold SpringHarbor, N.Y., 1989; Berger & Kimmel, Methods in Enzymology, Vol. 152:Guide to Molecular Cloning Techniques, Academic Press, Inc., San Diego,Calif., 1987; Co et al. (1992) J. Immunol., 148: 1149). For example,genes encoding heavy and light chains are cloned from a trioma's genomicDNA or cDNA produced by reverse transcription of the trioma's RNA.Cloning is accomplished by conventional techniques including the use ofPCR primers that hybridize to the sequences flanking or overlapping thegenes, or segments of genes, to be cloned.

[0128] Typically, recombinant constructs comprise DNA segments encodinga complete human immunoglobulin heavy chain and/or a complete humanimmunoglobulin light chain of an immunoglobulin expressed by a triomacell line. Alternatively, DNA segments encoding only a portion of theprimary antibody genes are produced, which portions possess bindingand/or effector activities. Other recombinant constructs containsegments of trioma cell line immunoglobulin genes fused to segments ofother immunoglobulin genes, particularly segments of other humanconstant region sequences (heavy and/or light chain). Human constantregion sequences can be selected from various reference sources,including but not limited to those listed in Kabat et al. (1987)Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services.

[0129] In addition to the DNA segments encoding anti-NBCCSimmunoglobulins or fragments thereof, other substantially homologousmodified immunoglobulins can be readily designed and manufacturedutilizing various recombinant DNA techniques known to those skilled inthe art such as site-directed mutagenesis (see Gillman & Smith (1979)Gene, 8: 81-97; Roberts et al. (1987) Nature 328: 731-734). Suchmodified segments will usually retain antigen binding capacity and/oreffector function. Moreover, the modified segments are usually not sofar changed from the original trioma genomic sequences to preventhybridization to these sequences under stringent conditions. Because,like many genes, immunoglobulin genes contain separate functionalregions, each having one or more distinct biological activities, thegenes may be fused to functional regions from other genes to producefusion proteins (e.g., immunotoxins) having novel properties or novelcombinations of properties.

[0130] The recombinant polynucleotide constructs will typically includean expression control sequence operably linked to the coding sequences,including naturally-associated or heterologous promoter regions.Preferably, the expression control sequences will be eukaryotic promotersystems in vectors capable of transforming or transfecting eukaryotichost cells. Once the vector has been incorporated into the appropriatehost, the host is maintained under conditions suitable for high levelexpression of the nucleotide sequences, and the collection andpurification of the human anti-NBCCS immunoglobulins.

[0131] These expression vectors are typically replicable in the hostorganisms either as episomes or as an integral part of the hostchromosomal DNA. Commonly, expression vectors will contain selectionmarkers, e.g., ampicillin-resistance or hygromycin-resistance, to permitdetection of those cells transformed with the desired DNA sequences.

[0132] In general, prokaryotes can be used for cloning the DNA sequencesencoding a human anti-NBCCS immunoglobulin chain. E. coli is oneprokaryotic host particularly useful for cloning the DNA sequences ofthe present invention. Microbes, such as yeast are also useful forexpression. Saccharomyces is a preferred yeast host, with suitablevectors having expression control sequences, an origin of replication,termination sequences and the like as desired. Typical promoters include3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeastpromoters include, among others, promoters from alcohol dehydrogenase 2,isocytochrome C, and enzymes responsible for maltose and galactoseutilization.

[0133] Mammalian cells are a particularly preferred host for expressingnucleotide segments encoding immunoglobulins or fragments thereof (see,e.g., Winnacker, From Genes to Clones, VCH Publishers, N.Y., 1987). Anumber of suitable host cell lines capable of secreting intactheterologous proteins have been developed in the art, and include CHOcell lines, various COS cell lines, HeLa cells, L cells and myeloma celllines. Preferably, the cells are nonhuman. Expression vectors for thesecells can include expression control sequences, such as an origin ofreplication, a promoter, an enhancer (Queen et al. (1986) Immunol. Rev.89: 49), and necessary processing information sites, such as ribosomebinding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Preferred expression controlsequences are promoters derived from endogenous genes, cytomegalovirus,SV40, adenovirus, bovine papillomavirus, and the like (see, e.g., Co etal. (1992) J. Immunol. 148: 1149).

[0134] The vectors containing the DNA segments of interest can betransferred into the host cell by well-known methods, depending on thetype of cellular host. For example, calcium chloride transfection iscommonly utilized for prokaryotic cells, whereas calcium phosphatetreatment, electroporation, lipofection, biolistics or viral-basedtransfection may be used for other cellular hosts. Other methods used totransform mammalian cells include the use of polybrene, protoplastfusion, liposomes, electroporation, and microinjection (see, generally,Sambrook et al., supra).

[0135] Once expressed, human anti-NBCCS immunoglobulins of the inventioncan be purified according to standard procedures of the art, includingHPLC purification, fraction column chromatography, gel electrophoresisand the like (see, generally, Scopes, Protein Purification,Springer-Verlag, NY, 1982). Detailed protocols for the production ofhuman antibodies can be found in U.S. Pat. No. 5,506,132.

[0136] Other approaches in vitro immunization of human blood. In thisapproach, human blood lymphocytes capable of producing human antibodiesare produced. Human peripheral blood is collected from the patient andis treated to recover mononuclear cells. The suppressor T-cells then areremoved and remaining cells are suspended in a tissue culture medium towhich is added the antigen and autologous serum and, preferably, anonspecific lymphocyte activator. The cells then are incubated for aperiod of time so that they produce the specific antibody desired. Thecells then can be fused to human myeloma cells to immortalize the cellline, thereby to permit continuous production of antibody (see U.S. Pat.No. 4,716,111).

[0137] In another approach, mouse-human hybridomas which produces humananti-NBCCS are prepared (see, e.g., U.S. Pat. No. 5,506,132). Otherapproaches include immunization of murines transformed to express humanimmunoglobulin genes, and phage display screening (Vaughan et al.supra.).

IV. Expression of NBCCS Polypeptides

[0138] A) De novo Chemical Synthesis.

[0139] The NBCCs proteins or subsequences thereof may be synthesizedusing standard chemical peptide synthesis techniques. Where the desiredsubsequences are relatively short (e.g., when a particular antigenicdeterminant is desired) the molecule may be synthesized as a singlecontiguous polypeptide. Where larger molecules are desired, subsequencescan be synthesized separately (in one or more units) and then fused bycondensation of the amino terminus of one molecule with the carboxylterminus of the other molecule thereby forming a peptide bond.

[0140] Solid phase synthesis in which the C-terminal amino acid of thesequence is attached to an insoluble support followed by sequentialaddition of the remaining amino acids in the sequence is the preferredmethod for the chemical synthesis of the polypeptides of this invention.Techniques for solid phase synthesis are described by Barany andMerrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides:Analysis, Synthesis, Biology. Vol 2: Special Methods in PeptideSynthesis, Part A., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156(1963), and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed PierceChem. Co., Rockford, Ill. (1984).

[0141] B) Recombinant Expression.

[0142] In a preferred embodiment, the NBCCS proteins or subsequencesthereof, are synthesized using recombinant DNA methodology. Generallythis involves creating a DNA sequence that encodes the fusion protein,placing the DNA in an expression cassette under the control of aparticular promoter, expressing the protein in a host, isolating theexpressed protein and, if required, renaturing the protein.

[0143] DNA encoding the NBCCS proteins or subsequences of this inventionmay be prepared by any suitable method as described above, including,for example, cloning and restriction of appropriate sequences or directchemical synthesis by methods such as the phosphotriester method ofNarang et al. Meth. Enzymol. 68: 90-99 (1979); the phosphodiester methodof Brown et al., Meth. Enzymol. 68: 109-151 (1979); thediethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859-1862 (1981); and the solid support method of U.S. Pat. No.4,458,066.

[0144] Chemical synthesis produces a single stranded oligonucleotide.This may be converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill would recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

[0145] Alternatively, subsequences may be cloned and the appropriatesubsequences cleaved using appropriate restriction enzymes. Thefragments may then be ligated to produce the desired DNA sequence.

[0146] In one embodiment, NBCCS proteins of this invention may be clonedusing DNA amplification methods such as polymerase chain reaction (PCR).Thus, for example, the nucleic acid sequence or subsequence is PCRamplified, using a sense primer containing one restriction site (e.g.,NdeI) and an antisense primer containing another restriction site (e.g.,HindIII). This will produce a nucleic acid encoding the desired NBCCSsequence or subsequence and having terminal restriction sites. Thisnucleic acid can then be easily ligated into a vector containing anucleic acid encoding the second molecule and having the appropriatecorresponding restriction sites. Suitable PCR primers can be determinedby one of skill in the art using the Sequence information provided inSEQ ID NO: 1. Appropriate restriction sites can also be added to thenucleic acid encoding the NBCCS protein or protein subsequence bysite-directed mutagenesis. The plasmid containing the NBCCS sequence orsubsequence is cleaved with the appropriate restriction endonuclease andthen ligated into the vector encoding the second molecule according tostandard methods.

[0147] The nucleic acid sequences encoding NBCCS proteins or proteinsubsequences may be expressed in a variety of host cells, including E.coli, other bacterial hosts, yeast, and various higher eukaryotic cellssuch as the COS, CHO and HeLa cells lines and myeloma cell lines. As theNBCCS proteins are typically found in eukaryotes, a eukaryote host ispreferred. The recombinant protein gene will be operably linked toappropriate expression control sequences for each host. For E. coli thisincludes a promoter such as the T7, trp, or lambda promoters, a ribosomebinding site and preferably a transcription termination signal. Foreukaryotic cells, the control sequences will include a promoter andpreferably an enhancer derived from immunoglobulin genes, SV40,cytomegalovirus, etc., and a polyadenylation sequence, and may includesplice donor and acceptor sequences.

[0148] The plasmids of the invention can be transferred into the chosenhost cell by well-known methods such as calcium chloride transformationfor E. coli and calcium phosphate treatment or electroporation formammalian cells. Cells transformed by the plasmids can be selected byresistance to antibiotics conferred by genes contained on the plasmids,such as the amp, gpt, neo and hyg genes.

[0149] Once expressed, the recombinant NBCCS proteins can be purifiedaccording to standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982), Deutscher, Methods inEnzymology Vol. 182: Guide to Protein Purification., Academic Press,Inc. N.Y. (1990)). Substantially pure compositions of at least about 90to 95% homogeneity are preferred, and 98 to 99% or more homogeneity aremost preferred. Once purified, partially or to homogeneity as desired,the polypeptides may then be used (e.g., as immunogens for antibodyproduction).

[0150] One of skill in the art would recognize that after chemicalsynthesis, biological expression, or purification, the NBCCS protein(s)may possess a conformation substantially different than the nativeconformations of the constituent polypeptides. In this case, it may benecessary to denature and reduce the polypeptide and then to cause thepolypeptide to re-fold into the preferred conformation. Methods ofreducing and denaturing proteins and inducing re-folding are well knownto those of skill in the art (See, Debinski et al. (1993) J. Biol.Chem., 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem., 4:581-585; and Buchner, et al., (1992) Anal. Biochem., 205: 263-270).Debinski et al., for example, describe the denaturation and reduction ofinclusion body proteins in guanidine-DTE. The protein is then refoldedin a redox buffer containing oxidized glutathione and L-arginine.

[0151] One of skill would recognize that modifications can be made tothe NBCCS proteins without diminishing their biological activity. Somemodifications may be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, a methionine added at the amino terminus to provide aninitiation site, or additional amino acids (e.g., poly His) placed oneither terminus to create conveniently located restriction sites ortermination codons or purification sequences.

V. Detection of NBCCS

[0152] As indicated above, abnormal (e.g., altered or deficient)expression of the human PTC gene is a causal factor in the developmentof basal cell carcinomas and, where the alteration is a heritablecharacter, in the etiology of nevoid basal cell carcinoma syndromeand/or the various developmental abnormalities characteristic of thissyndrome. It is believed that development of neoplasia requires completeinactivation of the NBCCS gene, however, partial inactivation creates apredisposition, either through haploinsufficiency or through increasedsusceptibility to complete inactivation (e.g., through a secondmutation), to basal cell carcinomas and/or NBCCS.

[0153] Thus, it is desirable to determine the presence or absence, orquantify, the expression of NBCCS polypeptides of the nucleic acidsencoding the NBCCS polypeptides. This may be accomplished by assayingthe gene product; NBCCS polypeptides themselves, or alternatively, byassaying the nucleic acids (DNA or mRNA) that encode the NBCCSpolypeptides. In particular, it is desirable to determine whether NBCCSexpression is present, absent, or abnormal (e.g. because of an abnormalgene product or because of abnormal expression levels as, for example,with a hemizygous gene). Particularly, where it is desired to determinea heritable propensity for abnormal NBCCS gene expression, it ispreferred to assay the host DNA for abnormal NBCCS genes or genetranscripts (mRNAs).

[0154] A) Sample Collection and Processing

[0155] The NBCCS (PTC) gene or gene product (i.e., mRNA or polypeptide)is preferably detected and/or quantified in a biological sample. As usedherein, a biological sample is a sample of biological tissue or fluidthat, in a healthy and/or pathological state, contains an NBCCS (PTC)nucleic acid or polypeptide. Such samples include, but are not limitedto, sputum, amniotic fluid, blood, blood cells (e.g., white cells),tissue or fine needle biopsy samples, urine, peritoneal fluid, andpleural fluid, or cells therefrom. Biological samples may also includesections of tissues such as frozen sections taken for histologicalpurposes. Although the sample is typically taken from a human patient,the assays can be used to detect NBCCS genes or gene products in samplesfrom any mammal, such as dogs, cats, sheep, cattle, and pigs.

[0156] The sample may be pretreated as necessary by dilution in anappropriate buffer solution or concentrated, if desired. Any of a numberof standard aqueous buffer solutions, employing one of a variety ofbuffers, such as phosphate, Tris, or the like, at physiological pH canbe used.

[0157] B) Nucleic Acid Assays.

[0158] In one embodiment, this invention provides for methods ofdetecting and/or quantifying human NBCCS (PTC) expression by assayingthe underlying NBCCS gene (or a fragment thereof) or by assaying theNBCCS gene transcript (mRNA). The assay can be for the presence orabsence of the normal gene or gene product, for the presence or absenceof an abnormal gene or gene product, or quantification of thetranscription levels of normal or abnormal NBCCS gene product.

[0159] i) Nucleic Acid Sample.

[0160] In a preferred embodiment, nucleic acid assays are performed witha sample of nucleic acid isolated from the organism to be tested. In thesimplest embodiment, such a nucleic acid sample is the total mRNAisolated from a biological sample. The nucleic acid (e.g., eithergenomic DNA or mRNA) may be isolated from the sample according to any ofa number of methods well known to those of skill in the art. One ofskill will appreciate that where alterations in the copy number of theNBCCS gene are to be detected genomic DNA is preferably isolated.Conversely, where expression levels of a gene or genes are to bedetected, preferably RNA (mRNA) is isolated.

[0161] Methods of isolating total DNA or mRNA are well known to those ofskill in the art. For example, methods of isolation and purification ofnucleic acids are described in detail in Chapter 3 of LaboratoryTechniques in Biochemistry and Molecular Biology: Hybridization WithNucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, P.Tijssen, ed. Elsevier, N.Y. (1993) and Chapter 3 of LaboratoryTechniques in Biochemistry and Molecular Biology: Hybridization WithNucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, P.Tijssen, ed. Elsevier, N.Y. (1993)).

[0162] In a preferred embodiment, the total nucleic acid is isolatedfrom a given sample using, for example, an acidguanidinium-phenol-chloroform extraction method and polyA⁺ mRNA isisolated by oligo dT column chromatography or by using (dT)n magneticbeads (see, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989), orCurrent Protocols in Molecular Biology, F. Ausubel et al., ed. GreenePublishing and Wiley-Interscience, New York (1987)).

[0163] Frequently, it is desirable to amplify the nucleic acid sampleprior to hybridization. One of skill in the art will appreciate thatwhatever amplification method is used, if a quantitative result isdesired, care must be taken to use a method that maintains or controlsfor the relative frequencies of the amplified nucleic acids.

[0164] Methods of “quantitative” amplification are well known to thoseof skill in the art. For example, quantitative PCR involvessimultaneously co-amplifying a known quantity of a control sequenceusing the same primers. This provides an internal standard that may beused to calibrate the PCR reaction. The high density array may theninclude probes specific to the internal standard for quantification ofthe amplified nucleic acid.

[0165] One preferred internal standard is a synthetic AW106 cRNA. TheAW106 cRNA is combined with RNA isolated from the sample according tostandard techniques known to those of skill in the art. The RNA is thenreverse transcribed using a reverse transcriptase to provide copy DNA.The cDNA sequences are then amplified (e.g., by PCR) using labeledprimers. The amplification products are separated, typically byelectrophoresis, and the amount of radioactivity (proportional to theamount of amplified product) is determined. The amount of mRNA in thesample is then calculated by comparison with the signal produced by theknown AW106 RNA standard. Detailed protocols for quantitative PCR areprovided in PCR Protocols, A Guide to Methods and Applications, Innis etal., Academic Press, Inc. N.Y., (1990).

[0166] Other suitable amplification methods include, but are not limitedto polymerase chain reaction (PCR) (Innis et al. (1990) PCR Protocols. Aguide to Methods and Application. Academic Press, Inc. San Diego),ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560,Landegren et al. (1988) Science 241: 1077, and Barringer et al. (1990)Gene 89: 117, transcription amplification (Kwoh et al. (1989) Proc.Natl. Acad. Sci. USA 86: 1173), and self-sustained sequence replication(Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA 87:1874).

[0167] ii) Hybridization Assays.

[0168] A variety of methods for specific DNA and RNA measurement usingnucleic acid hybridization techniques are known to those of skill in theart (see Sambrook et al. supra). For example, one method for evaluatingthe presence, absence, or quantity of DNA encoding NBCCS proteins in asample involves a Southern transfer. Briefly, the digested genomic DNAis run on agarose slab gels in buffer and transferred to membranes.

[0169] Hybridization is carried out using the nucleic acid probesspecific for the target NBCCS sequence or subsequence. Nucleic acidprobes are designed based on the nucleic acid sequences encoding NBCCCSproteins (see SEQ ID NO: 1). The probes can be full length or less thanthe full length of the nucleic acid sequence encoding the NBCCS protein.Shorter probes are empirically tested for specificity. Preferablynucleic acid probes are 20 bases or longer in length. (See Sambrook etal. for methods of selecting nucleic acid probe sequences for use innucleic acid hybridization.) Visualization of the hybridized portionsallows the qualitative determination of the presence or absence of DNAencoding NBCCS proteins.

[0170] Similarly, a Northern transfer may be used for the detection ofmRNA encoding NBCCS proteins. In brief, the mRNA is isolated from agiven cell sample using, for example, an acidguanidinium-phenol-chloroform extraction method. The mRNA is thenelectrophoresed to separate the mRNA species and the mRNA is transferredfrom the gel to a nitrocellulose membrane. As with the Southern blots,labeled probes are used to identify the presence or absence of NBCCSproteins.

[0171] A variety of nucleic acid hybridization formats are known tothose skilled in the art. For example, common formats include sandwichassays and competition or displacement assays. Hybridization techniquesare generally described in “Nucleic Acid Hybridization, A PracticalApproach,” Ed. Hames, B. D. and Higgins, S. J., IRL Press, (1985); Galland Pardue Proc. Natl. Acad. Sci. U.S.A. 63: 378-383 (1969); and John etal. Nature 223: 582-587 (1969).

[0172] For example, sandwich assays are commercially usefulhybridization assays for detecting or isolating nucleic acid sequences.Such assays utilize a “capture” nucleic acid covalently immobilized to asolid support and a labeled “signal” nucleic acid in solution. Theclinical sample will provide the target nucleic acid. The “capture”nucleic acid and “signal” nucleic acid probe hybridize with the targetnucleic acid to form a “sandwich” hybridization complex. To beeffective, the signal nucleic acid cannot hybridize with the capturenucleic acid.

[0173] Typically, labeled signal nucleic acids are used to detecthybridization. Complementary nucleic acids or signal nucleic acids maybe labeled by any one of several methods typically used to detect thepresence of hybridized polynucleotides. The most common method ofdetection is the use of autoradiography with ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P-labelled probes or the like. Other labels include ligands which bindto labeled antibodies, fluorophores, chemiluminescent agents, enzymes,and antibodies which can serve as specific binding pair members for alabeled ligand.

[0174] Detection of a hybridization complex may require the binding of asignal generating complex to a duplex of target and probepolynucleotides or nucleic acids. Typically, such binding occurs throughligand and anti-ligand interactions as between a ligand-conjugated probeand an anti-ligand conjugated with a signal.

[0175] The label may also allow indirect detection of the hybridizationcomplex. For example, where the label is a hapten or antigen, the samplecan be detected by using antibodies. In these systems, a signal isgenerated by attaching fluorescent or enzyme molecules to the antibodiesor, in some cases, by attachment to a radioactive label. (Tijssen, P.,“Practice and Theory of Enzyme Immunoassays,” Laboratory Techniques inBio-chemistry and Molecular Biology, Burdon, R. H., van Knippenberg, P.H., Eds., Elsevier (1985), pp. 9-20.).

[0176] The sensitivity of the hybridization assays may be enhancedthrough use of a nucleic acid amplification system which multiplies thetarget nucleic acid being detected. Examples of such systems include thepolymerase chain reaction (PCR) system and the ligase chain reaction(LCR) system. Other methods recently described in the art are thenucleic acid sequence based amplification (NASBA™, Cangene, Mississauga,Ontario) and Q Beta Replicase systems.

[0177] An alternative means for determining the level of expression of agene encoding an NBCCS protein is in situ hybridization. In situhybridization assays are well known and are generally described inAngerer, et al., Methods Enzymol., 152: 649-660 (1987). In an in situhybridization assay, cells or tissue specimens are fixed to a solidsupport, typically a glass slide. If DNA is to be probed, the cells aredenatured with heat or alkali. The cells are then contacted with ahybridization solution at a moderate temperature to permit annealing oflabeled probes specific to NBCCS proteins. The probes are preferablylabeled with radioisotopes or fluorescent reporters. Detection of NBCCSby in situ hybridization is detailed in Example 2.

[0178] iii) Amplification Based Assays.

[0179] In another embodiment, the NBCCS gene or gene product can bedetected (assayed) using an amplification based assay. In anamplification based assay, all or part of the NBCCS gene or transcript(e.g., mRNA or cDNA) is amplified and the amplification product is thendetected. Where there is no underlying gene or gene product to act as atemplate amplification is non-specific or non-existent and there is nosingle amplification product. Where the underlying gene or gene productis present, the target sequence is amplified providing an indication ofthe presence, absence, or quantity of he underlying gene or mRNA.

[0180] Amplification-based assays are well known to those of skill inthe art (see, e.g., Innis, supra.). The cDNA sequence provided for theNBCCS gene is sufficient to enable one of skill to routinely selectprimers to amplify any portion of the gene. In addition, Table 2provides primer pairs for the PCR amplification of each of the 21 exonscomprising the NBCCS (PTC) gene. Example 2, below, providesamplification protocols and illustrates detection of the NBCCS (PTC)gene using PCR.

[0181] Amplification primers can be selected to provide amplificationproducts that span specific deletions, truncations, and insertions, asdiscussed below (see, Section iv, below) thereby facilitating thedetection of specific abnormalities.

[0182] iv) Specific Detection of Abnormalities (eg., Mutations).

[0183] Abnormal NBCCS (PTC) genes or gene products are characterized bypremature stop codons, deletions, or insertions. Typical abnormal genes(PTC mutations) are illustrated in Tables 3, 5, 6, 7 and 9. Prematurestop codons and deletions can be detected by decreased size of the geneor gene product (mRNA transcript or cDNA). Similarly, insertions can bedetected by increased size of the gene or gene product. Alternatively,mutations can be determined by sequencing of the gene or gene productaccording to standard methods.

[0184] In addition, amplification assays and hybridization probes can beselected to specifically target particular abnormalities. For example,where the abnormality is a deletion, nucleic acid probes oramplification primers can be selected that specifically hybridize to oramplify, respectively, the nucleic acid sequence that is deleted in theabnormal gene. The probe will fail to hybridize, or the amplificationreaction will fail to provide specific amplification, to abnormalversions of the NBCCS (PTC) nucleic acids which have the deletion.Alternatively, the probe or amplification reaction can be designed tospan the entire deletion or either end of the deletion (deletionjunction). Similarly, probes and amplification primers can be selectedthat specifically target point mutations or insertions. Methods fordetecting specific mutations were described in, for example, U.S. Pat.No. 5,512,441. In the case of PCR, amplification primers can be designedto hybridize to a portion of the NBCCS (PTC) gene but the terminalnucleotide at the 3′ end of the primer can be used to discriminatebetween the mutant and wild-type forms of NBCCS (PTC) gene. If theterminal base matches the point mutation or the wild-type sequence,polymerase dependent extension can proceed and an amplification productis detected. This method for detecting point mutations or polymorphismswas described in detail by Sommer et al., (1989) Mayo Clin. Proc.64:1361-1372. By using appropriate controls, one can develop a kithaving both positive and negative amplification products. The productscan be detected using specific probes or by simply detecting theirpresence or absence. A variation of the PCR method uses LCR where thepoint of discrimination, i.e., either the point mutation or thewild-type bases fall between the LCR oligonucleotides. The ligation ofthe oligonucleotides becomes the means for discriminating between themutant and wild-type forms of the NBCCS (PTC) gene.

[0185] A variety of automated solid-phase detection techniques are alsoappropriate for detecting the presence or absence of mutations in theNBCCS (PTC) gene. For instance, very large scale immobilized polymerarrays (VLSIPS™), available from Affymetrix, Inc. in Santa Clara, Calif.are used for the detection of nucleic acids having specific sequences ofinterest. See, Fodor et al. (1991) Science, 251: 767- 777; Sheldon etal. (1993) Clinical Chemistry 39(4): 718-719, and Kozal et al. (1996)Nature Medicine 2(7): 753-759. For example, oligonucleotides thathybridize to all known NBCCS (PTC) mutations can be synthesized on a DNAchip (such chips are available from Affymetrix) and the nucleic acidsfrom samples hybridized to the chip for simultaneous analysis of thesample nucleic acid for the presence or absence of any of the knownNBCCS (PTC) mutations. Protocols for detecting mutations are alsodescribed in, for example, Tijssen (1993) Laboratory Techniques inbiochemistry and molecular biology—hybridization with nucleic acidprobes parts I and II, Elsevier, N.Y.,and Choo (ed) (1994) Methods InMolecular Biology Volume 33—In Situ Hybridization Protocols, HumanaPress Inc., New Jersey (see also, other books in the Methods inMolecular Biology series).

[0186] iv) Detection of Expression Levels.

[0187] Where it is desired to quantify the transcription level (andthereby expression) of a normal or mutated NBCCS genes in a sample, thenucleic acid sample is one in which the concentration of the mRNAtranscript(s) of the NBCCS gene, or the concentration of the nucleicacids derived from the mRNA transcript(s), is proportional to thetranscription level (and therefore expression level) of that gene.Similarly, it is preferred that the hybridization signal intensity beproportional to the amount of hybridized nucleic acid. While it ispreferred that the proportionality be relatively strict (e.g., adoubling in transcription rate results in a doubling in mRNA transcriptin the sample nucleic acid pool and a doubling in hybridization signal),one of skill will appreciate that the proportionality can be morerelaxed and even non-linear. Thus, for example, an assay where a 5 folddifference in concentration of the target mRNA results in a 3 to 6 folddifference in hybridization intensity is sufficient for most purposes.Where more precise quantification is required appropriate controls canbe run to correct for variations introduced in sample preparation andhybridization as described herein. In addition, serial dilutions of“standard” target mRNAs can be used to prepare calibration curvesaccording to methods well known to those of skill in the art. Of course,where simple detection of the presence or absence of a transcript isdesired, no elaborate control or calibration is required.

[0188] C) NBCCS Polypeptide Assays.

[0189] The expression of the human NBCCS (PTC) gene can also be detectedand/or quantified by detecting or quantifying the expressed NBCCSpolypeptide. The NBCCS polypeptides can be detected and quantified byany of a number of means well known to those of skill in the art. Thesemay include analytic biochemical methods such as electrophoresis,capillary electrophoresis, high performance liquid chromatography(HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography,and the like, or various immunological methods such as fluid or gelprecipitin reactions, immunodiffusion (single or double),inununoelectrophoresis, radioimmunoassay(RIA), enzyme-linkedimmunosorbent assays (ELISAs), immunofluorescent assays, westernblotting, and the like.

[0190] In a particularly preferred embodiment, the NBCCS polypeptidesare detected in an electrophoretic protein separation, more preferablyin a two-dimensional electrophoresis, while in a most preferredembodiment, the NBCCS polypeptides are detected using an immunoassay.

[0191] As used herein, an immunoassay is an assay that utilizes anantibody to specifically bind to the analyte (NBCCS polypeptide). Theimmunoassay is thus characterized by detection of specific binding of aNBCCS polypeptide to an anti-NBCCS antibody as opposed to the use ofother physical or chemical properties to isolate, target, and quantifythe analyte.

[0192] 1) Electrophoretic Assays.

[0193] As indicated above, the presence or absence of NBCCS polypeptidesin a biological sample can be determined using electrophoretic methods.Means of detecting proteins using electrophoretic techniques are wellknown to those of skill in the art (see generally, R. Scopes (1982)Protein Purification, Springer-Verlag, N.Y.; Deutscher, (1990) Methodsin Enzymology Vol. 182: Guide to Protein Purification., Academic Press,Inc., N.Y.).

[0194] 2) Immunological Binding Assays.

[0195] In a preferred embodiment, the NBCCS polypeptides are detectedand/or quantified using any of a number of well recognized immunologicalbinding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110;4,517,288; and 4,837,168). For a review of the general immunoassays, seealso Methods in Cell Biology Volume 37: Antibodies in Cell Biology,Asai, ed. Academic Press, Inc. New York (1993); Basic and ClinicalImmunology 7th Edition, Stites & Terr, eds. (1991). Immunologicalbinding assays (or immunoassays) typically utilize a “capture agent” tospecifically bind to and often immobilize the analyte (in this caseNBCCS polypeptide or subsequence). The capture agent is a moiety thatspecifically binds to the analyte. In a preferred embodiment, thecapture agent is an antibody that specifically binds NBCCSpolypeptide(s). The antibody (anti-NBCCS) may be produced by any of anumber of means well known to those of skill in the art as describedabove in Section III(A).

[0196] Immunoassays also often utilize a labeling agent to specificallybind to and label the binding complex formed by the capture agent andthe analyte. The labeling agent may itself be one of the moietiescomprising the antibody/analyte complex. Thus, the labeling agent may bea labeled NBCCS polypeptide or a labeled anti-NBCCS antibody.Alternatively, the labeling agent may be a third moiety, such as anotherantibody, that specifically binds to the antibody/NBCCS complex.

[0197] In a preferred embodiment, the labeling agent is a second humanNBCCS antibody bearing a label. Alternatively, the second NBCCS antibodymay lack a label, but it may, in turn, be bound by a labeled thirdantibody specific to antibodies of the species from which the secondantibody is derived. The second can be modified with a detectablemoiety, such as biotin, to which a third labeled molecule canspecifically bind, such as enzyme-labeled streptavidin.

[0198] Other proteins capable of specifically binding immunoglobulinconstant regions, such as protein A or protein G may also be used as thelabel agent. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (see,generally Kronval, et al. (1973) J. Immunol., 111: 1401-1406, andAkerstrom, et al. (1985) J. Immunol., 135: 2589-2542).

[0199] Throughout the assays, incubation and/or washing steps may berequired after each combination of reagents. Incubation steps can varyfrom about 5 seconds to several hours, preferably from about 5 minutesto about 24 hours. However, the incubation time will depend upon theassay format, analyte, volume of solution, concentrations, and the like.Usually, the assays will be carried out at ambient temperature, althoughthey can be conducted over a range of temperatures, such as 10° C. to40° C.

[0200] a) Non-Competitive Assay Formats.

[0201] Immunoassays for detecting NBCCS polypeptide may be eithercompetitive or noncompetitive. Noncompetitive immunoassays are assays inwhich the amount of captured analyte (in this case NBCCS) is directlymeasured. In one preferred “sandwich” assay, for example, the captureagent (anti-NBCCS antibodies) can be bound directly to a solid substratewhere they are immobilized. These immobilized antibodies then captureNBCCS present in the test sample. The NBCCS thus immobilized is thenbound by a labeling agent, such as a second human NBCCS antibody bearinga label. Alternatively, the second NBCCS antibody may lack a label, butit may, in turn, be bound by a labeled third antibody specific toantibodies of the species from which the second antibody is derived. Thesecond can be modified with a detectable moiety, such as biotin, towhich a third labeled molecule can specifically bind, such asenzyme-labeled streptavidin.

[0202] b) Competitive Assay Formats.

[0203] In competitive assays, the amount of analyte (NBCCS) present inthe sample is measured indirectly by measuring the amount of an added(exogenous) analyte (NBCCS) displaced (or competed away) from a captureagent (anti NBCCS antibody) by the analyte present in the sample. In onecompetitive assay, a known amount of, in this case, NBCCS is added tothe sample and the sample is then contacted with a capture agent, inthis case an antibody that specifically binds NBCCS. The amount of NBCCSbound to the antibody is inversely proportional to the concentration ofNBCCS present in the sample.

[0204] In a particularly preferred embodiment, the antibody isimmobilized on a solid substrate. The amount of NBCCS bound to theantibody may be determined either by measuring the amount of NBCCSpresent in an NBCCS/antibody complex, or alternatively by measuring theamount of remaining uncomplexed NBCCS. The amount of NBCCS may bedetected by providing a labeled NBCCS molecule.

[0205] A hapten inhibition assay is another preferred competitive assay.In this assay a known analyte, in this case NBCCS is immobilized on asolid substrate. A known amount of anti-NBCCS antibody is added to thesample, and the sample is then contacted with the immobilized NBCCS. Inthis case, the amount of anti-NBCCS antibody bound to the immobilizedNBCCS is inversely proportional to the amount of NBCCS present in thesample. Again the amount of immobilized antibody may be detected bydetecting either the immobilized fraction of antibody or the fraction ofthe antibody that remains in solution. Detection may be direct where theantibody is labeled or indirect by the subsequent addition of a labeledmoiety that specifically binds to the antibody as described above.

[0206] c) Other Assay Formats.

[0207] In a particularly preferred embodiment, Western blot (immunoblot)analysis is used to detect and quantify the presence of NBCCS in thesample. The technique generally comprises separating sample proteins bygel electrophoresis on the basis of molecular weight, transferring theseparated proteins to a suitable solid support, (such as anitrocellulose filter, a nylon filter, or derivatized nylon filter), andincubating the sample with the antibodies that specifically bind NBCCS.The anti-NBCCS antibodies specifically bind to NBCCS on the solidsupport. These antibodies may be directly labeled or alternatively maybe subsequently detected using labeled antibodies (e.g., labeled sheepanti-mouse antibodies) that specifically bind to the anti-NBCCS.

[0208] Other assay formats include liposome immunoassays (LIA), whichuse liposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see, Monroe et al.(1986) Amer. Clin. Prod. Rev. 5:34-41).

[0209] d) Scoring of the Assay.

[0210] The assays of this invention as scored (as positive or negativefor NBCCS polypeptide) according to standard methods well known to thoseof skill in the art. The particular method of scoring will depend on theassay format and choice of label. For example, a Western Blot assay canbe scored by visualizing the colored product produced by the enzymaticlabel. A clearly visible colored band or spot at the correct molecularweight is scored as a positive result, while the absence of a clearlyvisible spot or band is scored as a negative. In a preferred embodiment,a positive test will show a signal intensity (e.g., NBCCS polypeptidequantity) at least twice that of the background and/or control and morepreferably at least 3 times or even at least 5 times greater than thebackground and/or negative control.

[0211] e) Reduction of Non-specific Binding.

[0212] One of skill in the art will appreciate that it is oftendesirable to reduce non-specific binding in immunoassays. Particularly,where the assay involves an antigen or antibody immobilized on a solidsubstrate it is desirable to minimize the amount of non-specific bindingto the substrate. Means of reducing such non-specific binding are wellknown to those of skill in the art. Typically, this involves coating thesubstrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin are widely used with powdered milk being most preferred.

[0213] f) Labels.

[0214] The particular label or detectable group used in the assay is nota critical aspect of the invention, so long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g. Dynabeads™),fluorescent dyes (e.g., fluorescein isothiocyanate, texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic (e.g. polystyrene,polypropylene, latex, etc.) beads.

[0215] The label may be coupled directly or indirectly to the desiredcomponent of the assay according to methods well known in the art. Asindicated above, a wide variety of labels may be used, with the choiceof label depending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

[0216] Non-radioactive labels are often attached by indirect means.Generally, a ligand molecule (e.g., biotin) is covalently bound to themolecule. The ligand then binds to an anti-ligand (e.g., streptavidin)molecule which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, for example, biotin,thyroxine, and cortisol, it can be used in conjunction with the labeled,naturally occurring anti-ligands. Alternatively, any haptenic orantigenic compound can be used in combination with an antibody.

[0217] The molecules can also be conjugated directly to signalgenerating compounds, e.g., by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, oroxidoreductases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g, luminol. For a review of variouslabeling or signal producing systems which may be used, see, U.S. Pat.No. 4,391,904).

[0218] Means of detecting labels are well known to those of skill in theart. Thus, for example, where the label is a radioactive label, meansfor detection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple calorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

[0219] Some assay formats do not require the use of labeled components.For instance, agglutination assays can be used to detect the presence ofthe target antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

[0220] g) Substrates.

[0221] As mentioned above, depending upon the assay, various components,including the antigen, target antibody, or anti-human antibody, may bebound to a solid surface. Many methods for immobilizing biomolecules toa variety of solid surfaces are known in the art. For instance, thesolid surface may be a membrane (e.g., nitrocellulose), a microtiterdish (e.g., PVC, polypropylene, or polystyrene), a test tube (glass orplastic), a dipstick (e.g. glass, PVC, polypropylene, polystyrene,latex, and the like), a microcentrifuge tube, or a glass or plasticbead. The desired component may be covalently bound or noncovalentlyattached through nonspecific bonding.

[0222] A wide variety of organic and inorganic polymers, both naturaland synthetic may be employed as the material for the solid surface.Illustrative polymers include polyethylene, polypropylene,poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethyleneterephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidenedifluoride (PVDF), silicones, polyformaldehyde, cellulose, celluloseacetate, nitrocellulose, and the like. Other materials which may beemployed, include paper, glasses, ceramics, metals, metalloids,semiconductive materials, cements or the like. In addition, are includedsubstances that form gels, such as proteins (e.g., gelatins),lipopolysaccharides, silicates, agarose and polyacrylamides can be used.Polymers which form several aqueous phases, such as dextrans,polyalkylene glycols or surfactants, such as phospholipids, long chain(12-24 carbon atoms) alkyl ammonium salts and the like are alsosuitable. Where the solid surface is porous, various pore sizes may beemployed depending upon the nature of the system.

[0223] In preparing the surface, a plurality of different materials maybe employed, particularly as laminates, to obtain various properties.For example, protein coatings, such as gelatin can be used to avoidnon-specific binding, simplify covalent conjugation, enhance signaldetection or the like.

[0224] If covalent bonding between a compound and the surface isdesired, the surface will usually be polyfunctional or be capable ofbeing polyfunctionalized. Functional groups which may be present on thesurface and used for linking can include carboxylic acids, aldehydes,amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercaptogroups and the like. The manner of linking a wide variety of compoundsto various surfaces is well known and is amply illustrated in theliterature. See, for example, Immobilized Enzymes, Ichiro Chibata,Halsted Press, New York, 1978, and Cuatrecasas (1970) J. Biol. Chem. 2453059).

[0225] In addition to covalent bonding, various methods fornoncovalently binding an assay component can be used. Noncovalentbinding is typically nonspecific absorption of a compound to thesurface. Typically, the surface is blocked with a second compound toprevent nonspecific binding of labeled assay components. Alternatively,the surface is designed such that it nonspecifically binds one componentbut does not significantly bind another. For example, a surface bearinga lectin such as Concanavalin A will bind a carbohydrate containingcompound but not a labeled protein that lacks glycosylation. Varioussolid surfaces for use in noncovalent attachment of assay components arereviewed in U.S. Pat. Nos. 4,447,576 and 4,254,082.

[0226] C) Evaluation of NBCCS Expression Levels and/or AbnormalExpression.

[0227] One of skill will appreciate that abnormal expression levels orabnormal expression products (e.g., mutated transcripts, truncated ornon-sense polypeptides) are identified by comparison to normalexpression levels and normal expression products. Normal levels ofexpression or normal expression products can be determined for anyparticular population, subpopulation, or group of organisms according tostandard methods well known to those of skill in the art. Typically thisinvolves identifying healthy organisms (i.e. organisms without NBCCS orbasal cell carcinomas) and measuring expression levels of the NBCCS(PTC) gene (as described herein) or sequencing the gene, mRNA, orreverse transcribed cDNA, to obtain typical (normal) sequencevariations. Application of standard statistical methods used inmolecular genetics permits determination of baseline levels ofexpression, and normal gene products as well as significant deviationsfrom such baseline levels.

[0228] D) Detection Kits.

[0229] The present invention also provides for kits for the diagnosis oforganisms (e.g., patients) with a predisposition (at risk) nevoid basalcell carcinoma or for sporadic basal cell carcinomas. The kitspreferably include one or more reagents for determining the presence orabsence of the NBCCS gene, for quantifying expression of the NBCCS gene,or for detecting an abnormal NBCCS gene or expression products of anabnormal NBCCS gene. Preferred reagents include nucleic acid probes thatspecifically bind to the normal NBCCS gene, cDNA, or subsequencethereof, probes that specifically bind to abnormal NBCCS gene (e.g.,NBCCS containing premature truncations, insertions, or deletions),antibodies that specifically bind to normal NBCCS polypeptides orsubsequences thereof, or antibodies that specifically bind to abnormalNBCCS polypeptides or subsequences thereof. The antibody orhybridization probe may be free or immobilized on a solid support suchas a test tube, a microtiter plate, a dipstick and the like. The kit mayalso contain instructional materials teaching the use of the antibody orhybridization probe in an assay for the detection of a predispositionfor NBCCS.

[0230] The kits may include alternatively, or in combination with any ofthe other components described herein, an anti-NBCCS antibody. Theantibody can be monoclonal or polyclonal. The antibody can be conjugatedto another moiety such as a label and/or it can be immobilized on asolid support (substrate).

[0231] The kit(s) may also contain a second antibody for detection ofNBCCS polypeptide/antibody complexes or for detection of hybridizednucleic acid probes. The kit may contain appropriate reagents fordetection of labels, positive and negative controls, washing solutions,dilution buffers and the like.

VI. Modulation of Expression of Endozenous NBCCS Genes

[0232] In still another embodiment, this invention provides methods ofregulating the expression of endogenous NBCCS genes. The expression ofan NBCCS gene product may be increased as a method of preparingmitigating or eliminating the tumorigenic potential of a cell.Conversely, upregulation of NBCCS gene may induce neoplastictransformation and provide a convenient and controllable model systemfor the study of basal cell carcinomas.

[0233] Methods of altering the expression of endogenous genes are wellknown to those of skill in the art. Typically such methods involvealtering or replacing all or a portion of the regulatory sequencescontrolling expression of the particular gene that is to be regulated.In a preferred embodiment, the regulatory sequences (e.g., the nativepromoter) upstream of the NBCCS gene is altered.

[0234] This is typically accomplished by the use of homologousrecombination to introduce a heterologous nucleic acid into the nativeregulatory sequences. To downregulate expression the NBCCS gene product,simple mutations that either alter the reading frame or disrupt thepromoter are suitable. To upregulate expression of the NBCCS geneproduct, the native promoter(s) can be substituted with heterologouspromoter(s) that induce higher than normal levels of transcription.

[0235] In a particularly preferred embodiment, nucleic acid sequencescomprising the structural gene in question or upstream sequences areutilized for targeting heterologous recombination constructs. Upstreamand downstream sequences can be readily determined using the informationprovided herein. Such sequences, for example, can be extended using 5′-or 3′- RACE. and homologous recombination constructs created with onlyroutine experimentation.

[0236] The use of homologous recombination to alter expression ofendogenous genes is described in detail in U.S. Pat. No. 5,272,071, WO91/09955, WO 93/09222, WO 96/29411, WO 95/31560, and WO 91/12650.

VII. NBCCS (PTC) Therapeutics.

[0237] A) Pharmaceutical Compositions

[0238] The NBCCS polypeptides, NBCCS polypeptide subsequences,anti-NBCCS antibodies, and anti-NBCCS antibody-effector (e.g., enzyme,toxin, hormone, growth factor, drug, etc.) conjugates or fusion proteinsof this invention are useful for parenteral, topical, oral, or localadministration, such as by aerosol or transdermally, for prophylacticand/or therapeutic treatment. The pharmaceutical compositions can beadministered in a variety of unit dosage forms depending upon the methodof administration. For example, unit dosage forms suitable for oraladministration include powder, tablets, pills, capsules and lozenges. Itis recognized that the NBCCS polypeptides and related compoundsdescribed of, when administered orally, must be protected fromdigestion. This is typically accomplished either by complexing theprotein with a composition to render it resistant to acidic andenzymatic hydrolysis or by packaging the protein in an appropriatelyresistant carrier such as a liposome. Means of protecting proteins fromdigestion are well known in the art.

[0239] The pharmaceutical compositions of this invention areparticularly useful for topical administration to treat basal cellcarcinomas, or their precursors, solar keratoses. In another embodiment,the compositions are useful for parenteral administration, such asintravenous administration or administration into a body cavity or lumenof an organ. The compositions for administration will commonly comprisea solution of the NBCCS polypeptide, antibody or antibody chimera/fusiondissolved in a pharmaceutically acceptable carrier, preferably anaqueous carrier. A variety of aqueous carriers can be used, e.g.,buffered saline and the like. These solutions are sterile and generallyfree of undesirable matter. These compositions may be sterilized byconventional, well known sterilization techniques. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate and the like. The concentration of chimeric molecule in theseformulations can vary widely, and will be selected primarily based onfluid volumes, viscosities, body weight and the like in accordance withthe particular mode of administration selected and the patient's needs.

[0240] Thus, a typical pharmaceutical composition for intravenousadministration would be about 0.1 to 10 mg per patient per day. Dosagesfrom 0.1 up to about 100 mg per patient per day may be used,particularly when the drug is administered to a secluded site and notinto the blood stream, such as into a body cavity or into a lumen of anorgan. Substantially higher dosages are possible in topicaladministration. Actual methods for preparing parenterally administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in such publications as Remington'sPharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa.(1980).

[0241] The compositions containing the present NBCCS polypeptides,antibodies or antibody chimer/fusions, or a cocktail thereof (i.e., withother proteins), can be administered for therapeutic treatments. Intherapeutic applications, compositions are administered to a patientsuffering from a disease (e.g., NBCCS or basal cell carcinoma) in anamount sufficient to cure or at least partially arrest the disease andits complications. An amount adequate to accomplish this is defined as a“therapeutically effective dose.” Amounts effective for this use willdepend upon the severity of the disease and the general state of thepatient's health.

[0242] Single or multiple administrations of the compositions may beadministered depending on the dosage and frequency as required andtolerated by the patient. In any event, the composition should provide asufficient quantity of the proteins of this invention to effectivelytreat the patient.

[0243] Among various uses of the NBCCS polypeptides, polypeptidesubsequences, anti-NBCCS antibodies and anti-NBCCS-effectorchimeras/fusions of the present invention are included a variety ofdisease conditions caused by nevoid basal cell carcinoma syndrome and/orbasal cell carcinomas. Preferred applications include treatment ofNBCCS, in particular treatment of the developmental anomaliescharacteristic of NBCCS and treatment of cancers, in particular basalcell carcinomas.

[0244] B) Cellular Transformation and Gene Therapy.

[0245] The present invention provides packageable human NBCCS (PTC)nucleic acids (cDNAs) for the transformation of cells in vitro and invivo. These packageable nucleic acids can be inserted into any of anumber of well known vectors for the transfection and transformation oftarget cells and organisms as described below. The nucleic acids aretransfected into cells, ex vivo or in vivo, through the interaction ofthe vector and the target cell. The NBCCS cDNA, under the control of apromoter, then expresses the NBCCS protein thereby mitigating theeffects of absent NBCCS genes or partial inactivation of the NBCCS geneor abnormal expression of the NBCCS gene.

[0246] Such gene therapy procedures have been used to correct acquiredand inherited genetic defects, cancer, and viral infection in a numberof contexts. The ability to express artificial genes in humansfacilitates the prevention and/or cure of many important human diseases,including many diseases which are not amenable to treatment by othertherapies. As an example, in vivo expression of cholesterol-regulatinggenes, genes which selectively block the replication of HIV, andtumor-suppressing genes in human patients dramatically improves thetreatment of heart disease, AIDS, and cancer, respectively. For a reviewof gene therapy procedures, see Anderson, Science (1992) 256:808-813;Nabel and Felgner (1993) TIBTECH 11: 211-217; Mitani and Caskey (1993)TIBTECH 11: 162-166; Mulligan (1993) Science 926-932; Dillon (1993)TIBTECH 11: 167-175; Miller (1992) Nature 357: 455-460; Van Brunt (1988)Biotechnology 6(10): 1149-1154; Vigne (1995) Restorative Neurology andNeuroscience 8: 35-36; Kremer and Perricaudet (1995) British MedicalBulletin 51(1) 31-44; Haddada et al. (1995) in Current Topics inMicrobiology and Immunology Doerfler and Bohm (eds) Springer-Verlag,Heidelberg Germany; and Yu et al., Gene Therapy (1994) 1:13-26.

[0247] Delivery of the gene or genetic material into the cell is thefirst critical step in gene therapy treatment of disease. A large numberof delivery methods are well known to those of skill in the art. Suchmethods include, for example liposome-based gene delivery (Debs and Zhu(1993) WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques6(7): 682-691; Rose U.S. Pat. No. 5,279,833; Brigham (1991) WO 91/06309;and Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7414), andreplication-defective retroviral vectors harboring a therapeuticpolynucleotide sequence as part of the retroviral genome (see, e.g.,Miller et al. (1990) Mol. Cell. Biol. 10:4239 (1990); Kolberg (1992) J.NIH Res. 4:43, and Cometta et al. Hum. Gene Ther. 2:215 (1991)). Widelyused retroviral vectors include those based upon murine leukemia virus(MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus(SIV), human immuno deficiency virus (HIV), and combinations thereof.See, e.g., Buchscher et al. (1992) J. Virol. 66(5) 2731-2739; Johann etal. (1992) J. Virol. 66 (5):1635-1640 (1992); Sommerfelt et al., (1990)Virol. 176:58-59; Wilson et al. (1989) J. Virol. 63:2374-2378; Miller etal., J. Virol. 65:2220-2224 (1991); Wong-Staal et al., PCT/US94/05700,and Rosenburg and Fauci (1993) in Fundamental Immunology, Third EditionPaul (ed) Raven Press, Ltd., New York and the references therein, and Yuet al., Gene Therapy (1994) supra).

[0248] AAV-based vectors are also used to transduce cells with targetnucleic acids, e.g., in the in vitro production of nucleic acids andpeptides, and in in vivo and ex vivo gene therapy procedures. See, Westet al. (1987) Virology 160:38-47; Carter et al. (1989) U.S. Pat. No.4,797,368; Carter et al. WO 93/24641 (1993); Kotin (1994) Human GeneTherapy 5:793-801; Muzyczka (1994) J. Clin. Invest. 94:1351 and Samulski(supra) for an overview of AAV vectors. Construction of recombinant AAVvectors are described in a number of publications, including Lebkowski,U.S. Pat. No. 5,173,414; Tratschin et al. (1985)Mol. Cell. Biol.5(11):3251-3260; Tratschin, et al. (1984) Mol. Cell. Biol. 4:2072-2081;Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;McLaughlin et al. (1988) and Samulski et al. (1989) J. Virol.63:03822-3828. Cell lines that can be transformed by rAAV include thosedescribed in Lebkowski et al. (1988) Mol. Cell. Biol. 8: 3988-3996.

[0249] A) Ex vivo Transformation of Cells.

[0250] Ex vivo cell transformation for diagnostics, research, or forgene therapy (e.g., via re-infusion of the transformed cells into thehost organism) is well known to those of skill in the art. In apreferred embodiment, cells are isolated from the subject organism,transfected with the NBCCS gene or cDNA of this invention, andre-infused back into the subject organism (e.g., patient). Various celltypes suitable for ex vivo transformation are well known to those ofskill in the art. Particular preferred cells are progenitor or stemcells (see, e.g., Freshney et al., Culture of Animal Cells, a Manual ofBasic Technique, third edition Wiley-Liss, New York (1994)) and thereferences cited therein for a discussion of how to isolate and culturecells from patients).

[0251] As indicated above, in a preferred embodiment, the packageablenucleic acid encodes an NBCCS polypeptide under the control of anactivated or constitutive promoter. The transformed cell(s) expressfunctional NBCCS polypeptide which mitigates the effects of deficient orabnormal NBCCS gene expression.

[0252] In one particularly preferred embodiment, stem cells are used inex-vivo procedures for cell transformation and gene therapy. Theadvantage to using stem cells is that they can be differentiated intoother cell types in vitro, or can be introduced into a mammal (such asthe donor of the cells) where they will engraft in the bone marrow.Methods for differentiating CD34⁺ cells in vitro into clinicallyimportant immune cell types using cytokines such a GM-CSF, IFN-γ andTNF-α are known (see, Inaba et al. (1992) J. Exp. Med. 176: 1693-1702,and Szabolcs et al. (1995) 154: 5851-5861).

[0253] Stem cells are isolated for transduction and differentiationusing known methods. For example, in mice, bone marrow cells areisolated by sacrificing the mouse and cutting the leg bones with a pairof scissors. Stem cells are isolated from bone marrow cells by panningthe bone marrow cells with antibodies which bind unwanted cells, such asCD4⁺ and CD8⁺ (T cells), CD45⁺ (panB cells), GR-1 (granulocytes), andIa^(d) (differentiated antigen presenting cells). For an example of thisprotocol see, Inaba et al. (1992) J. Exp. Med. 176: 1693-1702.

[0254] In humans, bone marrow aspirations from iliac crests areperformed e.g., under general anesthesia in the operating room. The bonemarrow aspirations is approximately 1,000 ml in quantity and iscollected from the posterior iliac bones and crests. If the total numberof cells collected is less than about 2×10⁸/kg, a second aspirationusing the sternum and anterior iliac crests in addition to posteriorcrests is performed. During the operation, two units of irradiatedpacked red cells are administered to replace the volume of marrow takenby the aspiration. Human hematopoietic progenitor and stem cells arecharacterized by the presence of a CD34 surface membrane antigen. Thisantigen is used for purification, e.g., on affinity columns which bindCD34. After the bone marrow is harvested, the mononuclear cells areseparated from the other components by means of ficoll gradientcentrifugation. This is performed by a semi-automated method using acell separator (e.g., a Baxter Fenwal CS3000+ or Terumo machine). Thelight density cells, composed mostly of mononuclear cells are collectedand the cells are incubated in plastic flasks at 37° C. for 1.5 hours.The adherent cells (monocytes, macrophages and B-Cells) are discarded.The non-adherent cells are then collected and incubated with amonoclonal anti-CD34 antibody (e.g., the murine antibody 9C5) at 4° C.for 30 minutes with gentle rotation. The final concentration for theanti-CD34 antibody is 10 μg/ml. After two washes, paramagneticmicrospheres (DynaBeads, supplied by Baxter Immunotherapy Group, SantaAna, Calif.) coated with sheep antimouse IgG (Fe) antibody are added tothe cell suspension at a ratio of 2 cells/bead. After a furtherincubation period of 30 minutes at 4° C., the rosetted cells withmagnetic beads are collected with a magnet. Chymopapain (supplied byBaxter Immunotherapy Group, Santa Ana, Calif.) at a final concentrationof 200 U/ml is added to release the beads from the CD34+ cells.Alternatively, and preferably, an affinity column isolation procedurecan be used which binds to CD34, or to antibodies bound to CD34 (see,the examples below). See, Ho et al. (1995) Stem Cells 13 (suppl. 3):100-105. See also, Brenner (1993) Journal of Hematotherapy 2: 7-17.

[0255] In another embodiment, hematopoietic stem cells are isolated fromfetal cord blood. Yu et al. (1995) Proc. Natl. Acad. Sci. USA 92:699-703 describe a preferred method of transducing CD34⁺ cells fromhuman fetal cord blood using retroviral vectors.

[0256] B) In vivo Transformation

[0257] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.)containing therapeutic nucleic acids can be administered directly to theorganism for transduction of cells in vivo. Administration is by any ofthe routes normally used for introducing a molecule into ultimatecontact with blood or tissue cells. The packaged nucleic acids areadministered in any suitable manner, preferably with pharmaceuticallyacceptable carriers. Suitable methods of administering such packagednucleic acids are available and well known to those of skill in the art,and, although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

[0258] Pharmaceutically acceptable carriers are determined in part bythe particular composition being administered, as well as by theparticular method used to administer the composition. Accordingly, thereis a wide variety of suitable formulations of pharmaceuticalcompositions of the present invention.

[0259] Formulations suitable for oral administration can consist of (a)liquid solutions, such as an effective amount of the packaged nucleicacid suspended in diluents, such as water, saline or PEG 400; (b)capsules, sachets or tablets, each containing a predetermined amount ofthe active ingredient, as liquids, solids, granules or gelatin; (c)suspensions in an appropriate liquid; and (d) suitable emulsions. Tabletforms can include one or more of lactose, sucrose, mannitol, sorbitol,calcium phosphates, corn starch, potato starch, tragacanth,microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide,croscarmellose sodium, talc, magnesium stearate, stearic acid, and otherexcipients, colorants, fillers, binders, diluents, buffering agents,moistening agents, preservatives, flavoring agents, dyes, disintegratingagents, and pharmaceutically compatible carriers. Lozenge forms cancomprise the active ingredient in a flavor, usually sucrose and acaciaor tragacanth, as well as pastilles comprising the active ingredient inan inert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels, and the like containing, in addition to the activeingredient, carriers known in the art.

[0260] The packaged nucleic acids, alone or in combination with othersuitable components, can be made into aerosol formulations (i.e., theycan be “nebulized”) to be administered via inhalation. Aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

[0261] Suitable formulations for rectal administration include, forexample, suppositories, which consist of the packaged nucleic acid witha suppository base. Suitable suppository bases include natural orsynthetic triglycerides or paraffin hydrocarbons. In addition, it isalso possible to use gelatin rectal capsules which consist of acombination of the packaged nucleic acid with a base, including, forexample, liquid triglycerides, polyethylene glycols, and paraffinhydrocarbons.

[0262] Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, intraperitoneally,intravesically or intrathecally. Parenteral administration andintravenous administration are the preferred methods of administration.The formulations of packaged nucleic acid can be presented in unit-doseor multi-dose sealed containers, such as ampules and vials.

[0263] Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced by the packaged nucleic acid as described above in thecontext of ex vivo therapy can also be administered intravenously orparenterally as described above.

[0264] The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular vector employed and the condition of thepatient, as well as the body weight or surface area of the patient to betreated. The size of the dose also will be determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, or transduced cell type in aparticular patient.

[0265] In determining the effective amount of the vector to beadministered in the treatment or prophylaxis NBCCS predilection or onsetor basal cell carcinoma predilection or onset, the physician evaluatescirculating plasma levels of the vector, vector toxicities, progressionof the disease, and the production of anti-vector antibodies. Ingeneral, the dose equivalent of a naked nucleic acid from a vector isfrom about 1 μg to 100 μg for a typical 70 kilogram patient, and dosesof vectors which include a retroviral particle are calculated to yieldan equivalent amount of therapeutic nucleic acid.

[0266] For administration, inhibitors and transduced cells of thepresent invention can be administered at a rate determined by the LD-50of the inhibitor, vector, or transduced cell type, and the side-effectsof the inhibitor, vector or cell type at various concentrations, asapplied to the mass and overall health of the patient. Administrationcan be accomplished via single or divided doses.

[0267] In a preferred embodiment, prior to infusion, blood samples areobtained and saved for analysis. Between 1×10⁸ and 1×10¹² transducedcells are infused intravenously over 60- 200 minutes. Vital signs andoxygen saturation by pulse oximetry are closely monitored. Blood samplesare obtained 5 minutes and 1 hour following infusion and saved forsubsequent analysis. Leukopheresis, transduction and reinfusion can berepeated are repeated every 2 to 3 months. After the first treatment,infusions can be performed on a outpatient basis at the discretion ofthe clinician. If the reinfusion is given as an outpatient, theparticipant is monitored for at least 4, and preferably 8 hoursfollowing the therapy.

[0268] Transduced cells are prepared for reinfusion according toestablished methods. See, Abrahamsen et al. (1991) J. Clin. Apheresis,6: 48-53; Carter et al. (1988) J. Clin. Apheresis, 4:113-117; Aebersoldet al. (1988) J. Immunol. Meth., 112: 1-7; Muul et al. (1987) J.Immunol. Methods 101:171-181 and Carter etal. (1987) Transfusion 27:362-365. After a period of about 2-4 weeks in culture, the cells shouldnumber between 1×10⁸ and 1×10¹². In this regard, the growthcharacteristics of cells vary from patient to patient and from cell typeto cell type. About 72 hours prior to reinfusion of the transducedcells, an aliquot is taken for analysis of phenotype, and percentage ofcells expressing the therapeutic agent.

EXAMPLES

[0269] The following examples are offered to illustrate, but not tolimit the present invention.

Example 1 Cloning of a Human Patched Homologue

[0270] This example describes the isolation of a complete human PATCHEDcDNA sequence which encodes a putative protein of 1172 amino acids, anddisplays 61% sequence identity to the Drosophila PATCHED protein.Drosophila patched (ptc) is a segment polarity gene required for thecorrect patterning of larval segments and imaginal discs during flydevelopment (Nakano et al. (1989) Nature 341: 508-13; Hooper et al.(1989) Cell 59: 751-765). Based on genetic studies, patched is acomponent of the signaling pathway of the morphogen hedgehog (Basler etal. (1994) Nature 368: 208-214; Capdevila et al. (1994) EMBO J. 13:71-82; Ingham (1991) Nature 353: 184-187). Since patched is a putativemembrane-spanning protein, and is expressed in hedgehog responsivecells, it has been proposed to be the hedgehog receptor (Ingham (1991)supra.).

[0271] In vertebrates, several hedgehog homologs have been identified.The best characterized of them, sonic hedgehog, has been implicated inthe dorsal-ventral patterning of neural tube (Roelink etal. (1994) Cell76: 761-775; Roelink (1995) Cell 81: 445-455), in the differentiation ofsomites (Johnson et al. (1994) Cell 79: 1165-1173) and in theestablishing of the anterior-posterior axis of the limb bud (Riddle etal. (1993) Cell, 75: 1401-1416). The biochemical basis of hedgehogsignaling in vertebrates remains poorly understood and has been hamperedlargely by the lack of a proven receptor for the molecule.

Experimental Procedures

[0272] Cosmid Isolation

[0273] Cosmids used in this study were isolated from a human chromosome9-specific genomic cosmid library (LL09NCO1“P”, Biomedical SciencesDivision, Lawrence Livermore National Laboratory, Livermore, Calif.94550) by screening with the YAC clone ICI-2ef8 (UK Human Genome MappingProject Resource Centre). This clone contains the microsatellite markerD9S287 which has been localized to chromosome 9q²2.3 (Povey et al.(1994) Ann. Hum. Genet., 58: 177-250). The isolation of YAC DNA andhybridization was performed as described by Vorechovsky et al. (1994)Genomics, 21: 517-24. The localization of the cosmids was confirmed byhybridization to YAC ICI-2ef 8 resolved by means of pulse-field gelelectrophoresis. The 96 well plate format of the cosmid clones thatcontain PTC is 42H11, 96F9, 218A8, 226G7.

[0274] Library Screening

[0275] Human cDNA clones were isolated from a fetal brain cDNA libraryin the lambda ZAPII phage vector (Stratagene, La Jolla, Calif., USA),using standard procedures. The probes were labeled with [³²P]dCTP byrandom priming (Redisrime, Amersham). Positive clones were rescued usingthe 704 helper phage/pBluescript excision system (Rapid Excision Kit,Stratagene) and sequenced. Mouse genomic clones were isolated from a129SV lambda FixII library (Stratagene). Phage DNA was cut with EcoRIand hybridized with PTC specific probes. Mouse cDNA clones were isolatedfrom an 11.5 dpc mouse embryo (Swiss male) library constructed in lambdagt10. Hybridization was performed at 55° C. Positive clones weresubcloned into pBluescript II SK (Stratagene) digested with NotI.

[0276] Sequencing

[0277] Templates for sequencing were prepared from overnight cultures ofrescued cDNA clones and/or EcoRI cosmid fragments subcloned inpBluescript KS(+) using a plasmid purification kit (Qiagen). Sequencingwas performed with the Taq Dyedeoxy Terminator Cycle sequencing kit(Applied Biosystems) according to the manufacturer's instructions.Sequencing reactions were resolved on an ABI 373A automated sequencer.Sequence analysis was performed using the GCG software. BLAST searcheswere performed with the NCBI network service. PTC sequences have beendeposited in GENBANK under accession #U43148.

[0278] Northern Hybridization

[0279] Expression of human PTC mRNA was examined by Northernhybridization of human tissue blots (Clontech) using cDNA probes labeledwith [³²P]dCTP. Hybridization solution contained 5×SSPE, 10×Denhardt'ssolution, 100 mg/ml denatured, sheared herring sperm DNA, 50% formamideand 2% SDS. Washes were performed at 60° C. with 2×SSC and 0.1% SDS.

[0280] Chromosomal Localization

[0281] The chromosomal localization of human PTC was identified by PCRanalysis of DNA panels obtained from human-hamster hybrid cells. Thepanel consisted of both whole chromosome 9 hybrids and deletion hybridsof 9q22.3. The primers used were PTC1 (5′-TTG CAT AAC CAG CGA GTCT-3′(SEQ ID NO: 2)) and PTC2 (5′-CAA ATG TAC GAG CAC TTC AAGG-3′ (SEQ ID NO:3)). Murine Ptc was mapped by means of interspecific backcross mapping.The panels were provided by the Jackson Laboratory (Bar Harbor, Me.) andare the BSB panel from a cross (C57BL/6J×M spretus)×C57BL/6J and asimilar BSS panel made up of DNA from the reciprocal backcross(C57BL/6JEi×SPRET/Ei)×SPRET/Ei. Mapping was performed by means of SSCP(single strand conformation polymorphism) analysis with the primersW18F3 (5′-CTG TCA AGG TGA ATG GAC-3′ (SEQ ID NO: 4) and W18R3 (5′-GGGGTT ATT CTG TAA AAGG-3′ (SEQ ID NO: 5)). PCR reactions were performed inthe presence of [³²P]dCTP. The samples were resolved on a 6% acrylamidegel (2.6% cross-linking) at 4° C. at 70 watts within 1.5 hours. Geneticlinkage was performed by segregation analysis.

[0282] In situ Hybridization

[0283] Whole mount in situ hybridization on mouse embryos and subsequentsectioning was performed as described by Christiansen, et al. (1995)Mech. Dev. 51: 341-50. The mouse Ptc probe was a 706 bp NotI/PstI cDNAfragment from the 5′ end of the gene, subcloned in pBluescriptII SK. Theprobe was linearized with SacII, the overhang blunted by incubation with5 U/mg Klenow at 22° C. for 15 minutes, and antisense RNA synthesized bytranscribing with T7 RNA polymerase.

Results and Discussion

[0284] Cloning of a Human PTC Homolog

[0285] Cosmids used in this study were isolated from a human chromosome9-specific genomic cosmid library using the YAC clone ICI-2ef8. Thisclone contains the microsatellite marker D9S287 which has been localizedto chromosome 9q22.3. Sequencing of a 1.8 kb EcoRI fragment of cosmid42H11 yielded an open reading frame with significant homology to threeconsecutive stretches of the Drosophila ptc protein. Using the 1.8 kbEcoRI fragment as a probe the complete human and partial mouse PTC cDNAsequences were isolated.

[0286] The sequence of the human PTC cDNA consists of an open readingframe of 3888 nucleotides; also sequenced were 441 and 2240 nucleotideson the 5′ end and on the 3′end, respectively (SEQ ID NO: 1; FIG. 8). Theopen reading frame of human PTC cDNA encodes for a putative protein of1290 amino acids. This open reading frame is initiated by an ATG codonthat has a moderate match for the translational start consensus sequencein vertebrates (GAGGCTAUGT (SEQ ID NO: 6) in PTC versus GCCGCCAUGG (SEQID NO: 7) (Kozak (1991) J. Biol. Chem., 266: 19867-19870)). Assumingthat this codon encodes for the first amino acid of the protein, humanptc consists of 1296 amino acids with a relative molecular weight (Mr)of 131×103. It shows 61% sequence identity to its Drosophilacounterpart. Upstream of the ATG, the open reading frame extends foranother 354 nucleotides (starting at base pair 88 of the sequence shownin FIG. 8). The 3′ untranslated region contains a canonicalpolyadenylation signal (AATAAA (SEQ ID NO: 8)) as well as mRNAdestabilizing ATTTA (SEQ ID NO: 9) motifs. These are localized 1401nucleotides and 547, 743, and 1515 nucleotides after the terminationcodon, respectively.

[0287] An alternative transcript is observed that splices from exon 3 toexon 2a. The open reading frame ends in exon 2a (see, SEQ ID NO: 59) butdoes not contain an AUG. Exon 2 can splice to one of three differentfirst exons. Exon 1b (see SEQ ID NO: 58) is homologous to the describedfirst exon of the mouse mRNA and has an ATG followed by ORF. Exon 1a hasORF through the entire length and a potential splice acceptor site (see,e.g., SEQ ID No: 58). Exon 3 contains the first in-frame ATG for all thetranscripts except the one initiating in exon 1b. A map of the promoterregion of NBCCS (PC7) is provided in FIG. 3.

[0288] Hydropathy analysis (Kyte et al. (1982) J. Mol. Biol., 157:105-32) of the entire open reading frame of human PTC predicts thepresence of eight main hydrophobic stretches. Distribution of thehydrophobic blocks is remarkably well conserved between speciesindicating that human PTC, like its Drosophila counterpart, is anintegral membrane protein.

[0289] Chromosomal Localization of PTC Chromosomal localization of humanPTC on 9q22.3 was confirmed by PCR analysis of chromosome 9 hybrids, anddeletion hybrids of 9q22.3, human-hamster hybrid DNA panels. The primersused (PTC1, PTC2) were derived from a sequence of a 1.8 kb EcoRIfragment of cosmid 42H11. Primer PTC1 is derived from an exon sequenceand PTC2 from an intron sequence. All DNA hybridization and cDNAsequencing data suggest that human PTC is a single copy gene. Murine Ptcmaps to a short region of chromosome 13, close to the murine Facc locus(no recombination out of 188 meioses). This region contains the mousemutationsflexed tail (f) and purkinje cell degeneration (pcd), and it issyntenic with human 9q22-q31. Both f and pcd involve abnormaldevelopment of cells of the bone or brain and could be allelic to Ptc.

[0290] Expression of PTC

[0291] Northern blot analysis revealed five distinct PTC transcripts inall human tissues examined. Expression of these transcripts appears tobe differentially regulated. During mouse embryogenesis, expression ofPtc is first detected at E 8.0 dpc in ventral neuroepithelial tissue intwo separate domains along the midline. Expression persists in ventralneural cells through to 9.5 dpc and transcripts are also detected inlateral mesenchyme surrounding the neural tube. Ptc transcription isdetected in the somites soon after the time of their appearance andfollows a rostro-caudal gradient of expression. Somite expression isrestricted to epithelial cells within the medial aspects of each somite.Expression of Ptc is also detected in the posterior ectoderm of eachlimb bud from 10.0 dpc to 12.5 dpc. This region corresponds to surfaceectoderm that covers the ZPA. Other sites of Ptc expression during thisperiod include the inner surf aces of the branchial arches which flankthe oropharyngeal region, cells surrounding the placodes of thevibrissae and the genital eminence.

[0292] The expression pattern of Ptc points to a close relationshipbetween Ptc and the hedgehog family of morphogens. This relationship wasoriginally established in Drosophila (Ingham et al. (1991) Nature, 353:184-187). In vertebrates, the best characterized hedgehog homolog, sonichedgehog, has been implied in the induction of the Doorplate and motorneurons within the ventral neural tube (Jessell et al. (1990) HarveyLect., 86: 87-128; Yamada et al. (1993), 73: 673-686) as well as in thedifferentiation of sclerotome within the somites (Pourquie et al. (1993)Proc. Natl. Acad. Sci. USA, 90: 5242-5246). In the limb bud, sonichedgehog expression in the mesenchymal ‘zone of polarizing activity’triggers anters-posterior patterning of the limb (Riddle et al. (1993)Cell, 75: 1401-1416). Our data show that vertebrate PTC is expressed inall major target tissues of sonic hedgehog, such as the ventral neuraltube, somites and tissues surrounding the zone of polarizing activity ofthe limb bud. The striking spatial complementarity and temporalcoincidence of the sonic hedgehog and Ptc expression patterns suggestthat both genes might be members of a common signaling pathway.

[0293] The localization of PTC in the region containing the nevoid basalcell carcinoma syndrome (NBCCS) gene is intriguing. NBCCS is anautosomal dominant disorder which predisposes affected individuals tobasal cell carcinomas of the skin, medulloblastomas and various othertumors (Gorlin (1987) Medicine (Baltimore) 66: 98-113). Recent geneticstudies have placed the gene for the nevoid basal cell carcinomasyndrome to chromosome 9q22.3, between the markers Fanconi anaemiacomplementation group A (Farndon et al. (1994) Genomics, 23: 486-489)and D9S287 (Pericak-Vance (1995) Ann. Hum. Genet., 59: 347-365). Severallines of evidence suggest that PTC is a candidate gene for the nevoidbasal cell carcinoma syndrome. Ptc expression is compatible with thecongenital defects commonly found in NBCCS patients. Frequent symptomsin newborns and infants are developmental anomalies of the spine andribs (Gorlin (1987 supra.). These malformations could be due to a PTCdeficiency, expression of which coincides spatially and temporally withthe development of the neural tube and of the somites. In addition, Ptcexpression in the surface ectoderm surrounding the ZPA is consistentwith limb abnormalities often observed in the patients with NBCCS(Gorlin (1987 supra.). PTC expression in all adult tissues points to apleiotropic role of PTC in adult signal transduction pathways. Defectsin these signaling pathways could account for the symptoms which developpostnatally.

Example 2 Mutations of the Human Homologue of Drosophila Patched inNevoid Basal Cell Carcinoma Syndrome

[0294] The nevoid basal cell carcinoma syndrome (NBCCS) is an autosomaldominant disorder characterized by multiple basal cell carcinomas(BCCs), pits of the palms and soles, keratocysts of the jaw, and avariety of other tumors and developmental abnormalities. NBCCS wasmapped to chromosome 9q22.3 and both familial and sporadic BCCs displayloss of heterozygosity for markers in this region, consistent with thegene being a tumor suppressor. Example 1 describes the isolation of ahuman sequence (PTC) with strong, homology to the Drosophila segmentpolarity gene. This example shows that human PTC is expressed in many ofthe tissues affected in NBCCS patients. Single-stranded conformationpolymorphism analysis and sequencing revealed mutations of PTC inpatients with the syndrome and in related tumors. The data indicate thathuman PTC is also an NBCCS gene and that a reduction in expression ofthis gene leads to the developmental abnormalities observed in thesyndrome and that complete loss of patched function contributes totransformation of certain cell types.

Experimental Procedures

[0295] Subjects and Samples

[0296] DNA samples were collected from 363 individuals in 128 NBCCSkindreds. Patients were examined by a clinical geneticist, and diagnosisof Gorlin syndrome was based on at least two major features of thesyndrome; e.g., jaw cysts, palmar pits, multiple basal cell carcinomas,and a family history of typical Gorlin syndrome. Lymphoblastoid celllines were made from at least one affected member of 82 kindreds. 252basal cell carcinomas were collected as either fresh orparaffin-embedded specimens.

[0297] Short Tandem Repeat Polynmorphisms For linkage analysis and tumordeletion studies, PCR reactions were performed in 50 μL volumescontaining 100 ng of template DNA, 200 M dNTPs, 1.5 MM MgCl₂, 0.25 mMspermidine, 10 pM of each primer, I Ci ³²P dCTP (Amersham, ArlingtonHeights, Ill., USA), and 1.25 Units Taq polymerase (Promega, Madison,Wis., USA) in Promega buffer (10 mM TrisHCl, pH 9, 50 mM KCl, 0.1%Triton X-100). An Ericomp Dual Block thermocyler was set with thefollowing parameters for 25 cycles: 94° C., 1 min, 55° C., 30 sec, 72°C., 2 min. PCR products were analyzed on an 5% polyacrylamide gels.Autoradiography was carried out at −70° C. with Kodak XAR film. Loci inthe NBCCS region that were typed are shown in FIG. 1, and primersequences are available from the Genome Data Base(http://gdbwww.gdb.org).

[0298] Pulsed-Field Gel Electrophoresis (PFGE)

[0299] Cultured lymphoblastoid cells were embedded in LMP agarose (BioRad, Hercules Calif., USA) at a concentration of approximately2×10⁶/220- 1 block, and DNA was extracted according to standard methods(Sambrook et al. supra.) Quarter blocks were digested with SacII, MluI,NotI, eBssHI, NruI, and SfiI under conditions recommended by themanufacturer (New England Biolabs, Beverly, Mass., USA). Electrophoresiswas carried out with the Bio Rad CHEF DR 11 apparatus using, 1% agarosegels run for 20 hours at 200 volts with a pulse time of 75 sec. Forhigher resolution of fragments under 500 kb, a 25 second pulse time wasused.

[0300] Transfer to nylon membranes (Du Pont Gene Screen Plus, Du Pont,Co., Boston, Mass., USA) was performed according to the manufacturer'sinstruction after exposure of the gel to UV (6-7 mW/cm²) for twominutes. Probes were labeled to a specific activity of approximately 10⁹DPM/g, with dCT³²P (Amersham) by the random primed synthesis method(Boehringer Mannheim Kit, Boehringer Mannheim Corp., Indianapolis, Ind.,USA). Hybridization was carried out for 18 hours at 65° C. in 0.5 Msodium phosphate (pH 7.2), 7% SDS, 1% BSA, 1 mM EDTA and 200 μg/mlherring sperm. For probes containing repetitive sequences, sheared,sonicated human placental DNA (Sigma Chemical Co., St. Louis, Mo., USA)was added to the hybridization solution (500 μg/ml) and preassociatedwith the probe at 65° C. for 45 minutes prior to hybridization to thefilter. Filters were washed in 0.1×SSC with 1% SDS at 65° C. and exposedto autoradiographic film with an intensifying screen at −70° C. from 12hours to three days. Probes that detected similar sized fragments ondifferent blots were directly compared for comigrating fragments byhybridization to the same blot. Blots were stripped in 0.4N NAOH for 30min at room temperature between uses.

[0301] Fluorescence in situ Hybridization

[0302] Cosmid clones were labeled by nick translation withbiotin-11-dUTP, dioxigenin-11-dUTP, or both, and hybridized to metaphaseand interphase chromosomes under suppression conditions. Biotinylatedprobes were detected with 5 μg/ml of fluorescein isothiocyanate(FITC)-conjugated avidin DCS. Dioxigenin labeled probes were detectedwith 2 μg/ml anti-dioxigenin Fab conjugated to rhodamine. Thechromosomes were counterstained with 200 ng/ml of4,6-diamidino-2-phenylindole-dihydrochloride (DAPI). Images wereobtained using, a microscope coupled to a cooled CCD camera. Thedigitalized images were processed, pseudocolored and merged and thedistances between signals were measured.

[0303] Cosmid and BAC Screening

[0304] A gridded chromosome 9 cosmid library (LL09NCO1) was replicatedonto nylon filters (Gene Screen Dupont Plus, Du Pont Co.) and screenedaccording to the recommendations of the Human Genome Center, LawrenceLivermore National Laboratory. Positive coordinates were streaked out tosingle colonies and confirmed to contain the appropriate markers by PCRor hybridization. Gridded BAC filters were screened by hybridizationaccording to the manufacturer's recommendations (Research Genetics,Huntsville, Ala., USA). Because of the small chance of chimerism incosmids and BACS, fragments from the ends of contigs were mapped with apanel of human-hamster somatic cell hybrids to confirm theirlocalization on chromosome 9q22.

[0305] Isolation of cDNAs

[0306] Four methods were used to isolate candidate cDNAs. Direct cDNAselection (Parimoo et al. (1991) Proc. Natl. Acad. Sci., USA, 88:9623-9627) was applied to pools of cosmids and BACs. Following tworounds of selection, the PCR products were size fractionated and clonedinto PCRII (Invitrogen, Leek NV, Netherlands). Transformants weregridded into 96 well plates, and replica filters were probed with thegenomic template DNA to identify cDNAs that hybridized the correctgenomic region.

[0307] Exon trapping, was performed using the method developed byBuckler et al. (1991) Proc. Natl. Acad. Sci. USA, 88: 4005-4009, andlater modified by Church et al. (1994) Nature Genetics, 6: 98.BamHl/GblII digests of pools of 5 or 6 cosmids were cloned into theBamHl site of the splicing vector pSPL3b (Burn et al. (1995) Gene, 161:183-187). Trapped DNAs were sequenced and mapped back to the NBCCScandidate region by hybridization to the cosmids from which they werederived.

[0308] For HTF island cloning, YACs were size fractionated by pulsedfield gel electrophoresis, excised from the gel, and digested withBssHII. Subsequently vectorette linkers were added and PCR amplificationwas performed using a vectorette primer and a 5′ Alu primer (Valdes etal. (1994) Proc. Natl. Acad. Sci., 91: 5377-5381). After an initialdenaturation at 1 00° C. for 5 min, 30 amplification cycles wereperformed with denaturation for 1 min at 98° C., annealing for 1 min at60° C., and extension for 3 min at 72° C. Ten units Taq polymerase wereused in a total volume of 100 μl consisting of 50 mM KCl, 10 mM Tris pH9, 2 mM MgCl₂, 0.1% Triton and 200 μM dNTP. The PCR products wereelectrophoresed on a 1% agarose gel, in order to determine their size,and subsequently cloned into the PGEM T vector (Promega) by a shotgunprocedure.

[0309] For sequence sampling, the ends of chromosome 9 specific cosmidsor cosmid subclones were directly sequenced (Smith et al. (1994) NatureGenetics 7: 40-47. Sequencing was performed on an ABI 373 DNA sequencer.The resulting, end sequences were manually trimmed, examined for simplesequence repeats, and used to search the DNA sequence databases. Bothnucleotide and amino acid searches were performed. In addition sequenceswere examined for potential coding regions by GRAIL (Uberbacher andMural (1991) Proc. Natl. Acad. Sci. USA 88: 11261-11265.

[0310] Short cDNA fragments obtained by the methods outlined above wereextended by screening brain or epidermal cDNA libraries and by rapidamplification of cDNA ends (Marathon kit, Clontech, Palo Alto, Calif.,USA).

[0311] Intronlexon Structure of the Human Patched Gene

[0312] Oligonucleotides were chosen at approximately 150 bp intervalsspanning the cDNA of the human patched gene. PCR products were generatedfrom cosmids 226G7, 42H11, 55A16, or 96F9. Reactions were performed in a50 μl volume containing 25 pmol of various oligonucleotide combinations,200 μmol dNTPs, 1.5 mM, or 1.85 mM, or 2.2 mM MgCl₂, 5 U Taq polymerase,and amplified for 35 cycles of 94° C. for 30 s, 55° C. for 30 s 72° C.for 2.5 min. Some samples were amplified by long range PCR using theExpand Long Template PCR system (Boehringer Mannheim) according to themanufacturer's instructions. PCR products were resolved on a 1% agarosegel and isolated by a DNA purification kit (Jetsorb, Genomed, BadOeynhausen, Germany). Sequencing of PCR fragments was performed with theTaq Dyedeoxy Terminator Cycle Sequencing, kit (Applied Biosystems,Foster City, Calif., USA). Sequencing, reactions were resolved on an ABI373A automated sequencer. Positions of introns have been determined bypredicted splice donor or splice acceptor sites.

[0313] Mutation Detection

[0314] A combined SSCP (Orita et al. (1989) Ann. Hum. Genet. 59:347-365) and heteroduplex analysis (White et al. (1992) Genomics, 12:301-306) approach was used using optimized conditions (Glavac and Dean(1993) Hum. Mutation, 2: 404-414). DNA samples (100 ng) were amplifiedin PCR buffer containing, 1.5 mM MgCl, and ³²P-dCTP for 35 cycles of 94°C., 30 sec, 55° C., 30 sec, 72° C., 30 sec. Products were diluted 1:3 insolution, denatured at 95° C. for 2 min and 3 μl loaded directly ongels. Gel formulations used were 1) 6% acrylamide:Bis (2.6%crosslinking,), 10% glycerol, room temp, 45W; 2) 6% acrylamide:Bis (2.6%crosslinking), 4 60W; 3) 10% acrylamide:Bis (1.3% crosslinking) 10%glycerol4, 60W; 4) 0.5×MDE (ATGC Corp, Malvern, Pa.), 10% glycerol4,50W. Gels were run for 3-16 hours(3000Vh/100 bp), dried and exposed toX-ray film for 2-24 hrs. Heteroduplexes were identified from thedouble-stranded DNA at the bottom of the gels, and SSCPs from thesingle-stranded region.

[0315] Samples showing, variation were compared to other family membersto assess segregation of the alleles, or to normal DNA from the samepatient, in the case of tumors. PCR products with SSCP or heteroduplexvariants were treated with shrimp alkaline phosphatase and exonuclease I(United States Biochemical) and cycle sequenced with AmplitaqFS™ (PerkinElmer, Norwalk, Conn., USA). The products were analyzed on an AppliedBiosystems model 373 DNA sequencer.

Results and Discussion

[0316] Fine Mapping by Linkage and Tumor Deletion Studies

[0317] Since the original mapping of the gene in 1992, linkage studieshave narrowed the NBCCS region to a 4 cM interval between D9S180 andD9S196 (Goldstein et al. (1994) supra; Wicking et al. (1994) Genomics,22: 505-511). Famdon et al. (1994) supra, reported recombinationinvolving an unaffected individual that tentatively placed the geneproximal to D9S287.

[0318] The present experiments identified one recombination betweenD9S287 and FACC in a three-generation family. The recombinant individualwas a 1.5 year old female presumed to be affected on the basis ofmacrocephaly, strabismus, and frontal bossing. Some of the key featuresof the syndrome such as basal cell carcinomas, jaw cysts, and palmarpits, were lacking; but these features have age-dependent expression,and their presence in a young, child would not be expected. With theassumption that she carried the gene, the recombination in this familyplaced NBCCS proximal to D9S287.

[0319] Allelic loss in BCCs was concordant with linkage mapping inplacing, the gene between D9S 196 and D9S 180. Most hereditary tumorswith allelic loss deleted the entire region between the flanking,markers. However, one hereditary cardiac fibroma showed loss at D9S287but not D9S280 on the non-disease carrying, allele suggesting that thegene is located distal to D9S280. In sporadic BCCs four tumors werefound that retained D9S287 and lost more distal markers but also twotumors that lost the proximal marker D9S280, but not D9S287.

[0320] Several hypotheses can be proposed to explain this discrepancy intumor deletion mapping, and between tumor deletions and linkage studies.A gene other than NBCCS could be responsible for the allelic loss insome sporadic tumors. NBCCS is almost certainly the target of allelicloss in hereditary tumors because these tumors always lose the copy ofthe NBCCS gene from the unaffected parent and retain the inheritedmutation (Bonifas et al. (1994) Hum. Mol. Genet., 3: 447-448). If asecond locus were driving allelic loss, then the alleles from theaffected parent and the unaffected parent would be lost with equalfrequency. However, there may be two different tumor suppressors onchromosome 9q that are both important in basal cell carcinomas. The APCgene and MCC gene, for example, are both mutated in colon cancer and liewithin 1 Mb of each other on chromosome 5q (Hampton et al. (1992) Proc.Natl. Acad. Sci. USA 89: 8249-8253). The observation of a clearlydistinct pattern of allelic loss, involving, D9S180 but not D9S287, insquamous cell carcinoma of the skin supports the presence of more thanone tumor suppressor in the 9q22.3 region. Additionally a putative tumorsuppressor has been mapped to 9q21-3 1 in bladder cancers, and may bedistinct from the NBCCS gene (Knowles et al. (1995) Br. J. Urol. 75:57-66).

[0321] A second possibility is that regions on both sides of D9S287 aredeleted in some tumors, but that the more proximal deletions are notalways detected by available markers. In fact two tumors were observedthat deleted markers both proximal and distal to D9S127, probablyreflecting genetic instability in tumor cells. Finally, NBCCS could be alarge gene that extends on both sides of the D9S287 locus. Takentogether the data provided herein suggested that the most likelylocation of the NBCCS gene was between markers D9S280 and D9S287.Nevertheless, due to the discrepancies in tumor deletions, physicalmapping and cDNA isolation from the entire region between D9S196 andD9S180 were undertaken.

[0322] Phiysical Mapping

[0323] Twenty-nine YACs containing markers from this region wereobtained from the CEPH mega YAC library. Eighteen formed an overlappingcontig between D9S196 and D9S180 with at least 2-fold redundancy. Basedon this contig the minimum distance between the flanking markers was 1.5Mb, but virtually all large YACs had internal deletions as judged by STScontent. Additional YACs were obtained from the ICI library to provideredundancy in areas apparently prone to deletion. Cosmid and BAC contigswere constructed around known STSs and genes, and additional cosmidsfrom the region were isolated by hybridizing YACs to the LawrenceLivermore gridded cosmid library. In total over 800 cosmids specific tothis region were gridded into 96-well grid plates and contigs of BACS,P1s and cosmids covering 1.5 Mb were constructed (FIG. 1). Because ofdeletions in YACs and some gaps in the cosmid and BAC contig, pulsedfield gel electrophoresis (PFGE) and FISH were used to integrate thecloned regions. Based on the sizes of restriction fragments in thisregion and FISH estimates, the physical distance from D9S180 to D9S196was estimated at not less than 2 Mb.

[0324] Isolation of cDNAs

[0325] Harshman et al. (1995), supra, showed that different methods ofidentifying cDNAs from a genomic region result in a surprisinglydifferent array of candidate genes. Several methods were used to findgenes that map to chromosome 9q22 including sample sequencing ofcosmids, exon trapping, HTF island cloning, and direct selection ofcDNAs from BACs and cosmids. In addition, genes known to lie in thisgeneral area were more finely mapped by use of somatic cell hybrids madefrom two NBCCS patients with visible 9q22 deletions (submitted to NIGMSrepository), YAC contigs, and FISH. Ten genes, ten ESTs with sequencesin GENBANK, and 31 anonymous selected cDNA fragments, HTF island clones,and trapped exons with no known homology were identified (Table 1).

[0326] Screening Patients for Germline Deletions or Rearrangements

[0327] Because chromosome 9q22 appeared to be very gene rich, an attemptwas made to localize the NBCCS gene more precisely by searching forsubmicroscopic rearrangements in patients. Fifteen cosmids atapproximately 100-200 kb intervals spanning the region between D9S196and D9S180 were hybridized to PFGE blots of 82 unrelated NBCCS patients.In addition probes from genes known to map to the interval as well asthose identified in the course of the study were included in thisanalysis. PFGE variants were identified in three patients with genomicprobes from within the Fanconi's anemia complementation group C (FACC)gene. All three were heterozygous for SacII bands approximately 30 kbshorter than normal (310 vs 280 kb). The limit of resolution of PFGE wasabout 10 kb, so that it was not possible to determine whether theapparently identical variant SacII bands were exactly the same size.Other restriction enzymes including NotI, BssHII, MluI, SfiI, and NruIdid not show variant bands. The variations were not consistent withgermline deletions in these patients but could conceivably be caused bypoint mutations creating new restriction sites or other smallalterations such as the recurrent inversions seen in the F8C gene ofmany hemophiliacs (Lakich et al. (1993) Nat. Genetics, 5: 236-241).

[0328] The nature of the DNA alterations causing these changes on PFGEhas not yet been elucidated, but the data relating, them to the diseaseare compelling. The families of two of the patients with variations werenot available for study. However the third patient was a sporadic caseof NBCCS, and neither parent had the SacII alteration. The finding ofthis variant in a patient but not in her parents could be interpreted asthe result of hypermutability of some CG-rich region near FACC, but novariation in this region was identified in PFGE blots of over 100 normalchromosomes. TABLE 1 cDNA clones from the NBCCS region on chromosome9q22.3. Clone Designation^(a) Clone type^(b) cDNAs previously mapped tochromosome 9q and more finely mapped with somatic cell hybrids, PFGE,and YAC contigs. FACC Gene NCBP Gene HSD17B3 Gene TMOD Gene XPA Gene SYKGene WI-11139 EST (R14225) WI-11414 EST (T88697) WI-8684 EST (R14413)D9S1697 EST (R06574) D9S1145 EST (contains R17127 and Z38405) Novelclones or clones not previously mapped to chromosome 9q identified bysample sequencing, exon trapping, HTF island cloning or cDNA selection.ZNF169 Gene FBP1 Gene PTC Gene Coronin homologue Gene 2F1a EST (R39928)2F1b EST (T11435) 11F21 EST (Z43835) 31F3 EST (R16281) yo20g05.sl EST(Merck EST) plus 31 anonymous selected cDNA fragments, HTF islandclones, and trapped exons from YACs and cosmid pools^(c)

[0329] Evaluation of PTC as a Candidate Gene

[0330] Because variant PFGE bands were identified in the FACC region andone recombinant as well as tumor deletion studies suggested a possiblelocation near this marker, candidate cDNAs that mapped to this area wereexamined. FACC, itself, was not considered as a candidate becauseheterozygous mutations in this gene do not cause NBCCS (Strathdee et al.(1992) Nature, 356: 763-767). FACC and PTC (a novel human gene withstrong homology to Drosophila patched) hybridized to the same 650 kbNotI fragment and 675 kb and 1000 kb (partial) MluI fragments. Mouseinterspecies backcross analysis determined that there were norecombinants between the PTC and FACC genes out of 190 meioses. PTC andD9S287 were both present on ICI YAC 2EF8 having, a size of 350 kbstrongly suggesting, that PTC lies between D9S287 and FACC.

[0331] To screen for mutations in the PTC gene, the intron/exonboundaries of the gene were determined from genomic clones and longrange PCR products. PTC consists of 21 exons and the gene spansapproximately 34 kb (FIG. 2). Panels of unrelated NBCCS patients andBCCs were screened by single-stranded conformation polymorphism (SSCP)analysis (primers used for amplification of PTC exons are shown in Table2). Patients displaying variations were compared to unaffectedindividuals of the same race, and variants found only in affectedindividuals were further characterized by DNA sequencing. Of themutations identified in unrelated patients, four were deletions orinsertions resulting, in frameshifts and two were point mutationsleading, to premature stops (Table 3, FIGS. 4 and 5). An additionalfinding, confirming the relationship between mutations in PTC and thedisease was identification of a frameshift mutation in a sporadic NBCCSpatient that was not present in either of her unaffected parents (FIG.5).

[0332] To analyze the role of PTC in neoplasia, tumors related to thesyndrome were screened for mutations. Two sporadic basal cell carcinomaswith allelic loss of the NBCCS region had inactivating, mutations of theremaining allele (FIGS. 6 and 7). A tumor removed from the cheek had aCC to TT alteration, typical of UVB mutagenesis. The second tumor fromthe nose had a 14 bp deletion, a mutation that cannot be related to anyspecific environmental accent. Mutations have not yet been identified inany sporadic BCC not showing allelic loss of chromosome 9q22, andalternative modes of pathogenesis may be operative in these neoplasms.TABLE 2 Primers to amplify PTC exons. Exon Size Primer SEQ ID ExonPosition^(a) (bp) Name Primers^(b) NO  1   1-189 189 PTCF18 GAAGG CGAGCACCCA GAC 10 PTCR18 TCTTT CCCTC CTCTC CCTTC 11  1A alternate first >239PTCF22 GCTAT GGAAA TGCGT CGG 12 exon PTCR22 CAGTC CTGCT CTGTC CATCA 13 2  190-382 193 PTCF19 GTGGC TGAGA GCGAA GTTC 14 PTCR19 TTCCA CCCACAGCTC CTC 15  3  383-782 200 PTCF27 CTATT GTGTA TCCAA TGGCA GG 16 PTCR27ATTAG TAGGT GGACG CGGC 17  4  583-642 60 PTCF20 AGAG AA AT TTTT GTCTCTGC TTTT CA 18 PTCR20 CCTGA TCCAT GTAAC CTGTT TC 19  5  643-734 92PTCF21 GCAAA AATTT CTCAG GAACACC 20 PTCR21 TGGAA CAAAC AATGA TAAGCAA 21 6  735-933 199 PTCF15 CCTAC AAGGT GGATG CAGTG 22 18R2 TTTGC TCTCC ACCCTTCTGA 23  7  934-1055 122 11e18F GTGAC CTGCC TACTA ATTCCC 24 18R3 GGCTAGCGAG GATAA CGGTTTA 25  8 1056-1203 148 PTCF2 GAGGC AGTGG AAACT GCTTC 26PTCR2 TTGCA TAACC AGCGA GTCTG 27  9 1204-1492 288 PTCF23 GTGCT GTCGAGGCTT GTG 28 PTCR23 ACGGA CAGCA GATAA ATGGC 29 10 1493-1591 97 PTCF5GTGTT AGGTG CTGGT GGCA 30 PTCR5 CTTAG GAACA GAGGA AGCTG 31 11 1591-1835245 PTCF24PT TCTGC CACGT ATCTG CTCAC 32 CR24 CATGC TGAGA ATTGC AGGAA 3312 1836-2238 403 PTCF16 GGCCT ACACC GACAC ACAC 34 PTCR16 TTTTT TTGAAGACAG GAAGA GCC 35 PTC13R GTCAG CAGAC TGATT CAGGT 36 PTC37R AAGAT GAGAGTGTCC ACTTCG 37 13 2239-2548 310 PTCF14 GACAG CTTCT CTTTG TCCAG 38PTCR14 ACGCA AAAGA CCGAA AGGAC GA 39 14 2549-2691 143 PTCF13 AGGGT CCTTCTGGCT GCGAG 40 PTCR13 TCAGT GCCCA GCAGC TGGAG TA 41 15 2692-2875 185PTCF7 AACCC CATTC TCAAA GGCCT CTGTTC 42 PTCR7 CACCT CTGTA AGTTC CCAGACCT 43 16 2876-3156 281 PTCF12 AACTG TGATG CTCTT CTACC CTGG 44 PTCR12AAACT TCCCG GCTGC AGAAA GA 45 17 3157-3294 138 PTCF8 TTTGA TCTGA ACCGAGGACACC 46 PTCR8 CAAAC AGAGC CAGAG GAAATGG 47 18 3295-3437 143 PTCF11TAGGA CAGAG CTGAG CATTT ACC 48 PTC21R TACCT GACAA TGAAG TCG 49 193437-3537 101 PTCF11 TAGGA CAGAG CTGAG CATTT ACC 50 PTC21R TACCT GACAATGAAG TCG 51 20 3538-3792 255 PTCF25 AACAG AGGCC CCTGA AAAAT 52 PTCR25GATCA CTTGG TGGGC AGG 53 21 3793-4330^(c) 537 PTCF10 TCTAA CCCAC CCTCACCCTT 54 PTC31R ATTGT TAGGG CCAGA ATGCC 55 PTCF26 AGAAA AGGCT TGTGG CCAC56 PTCR26 TCACC CTCAG TTGGA GCTG 57

[0333] TABLE 3 Mutations in the PTC gene. Type of Sample TypeInheritance Exon Mutation Designation NBCCS F 5 premature stop C1081TNBCCS F 6 37 bp deletion del 804-840 NBCCS F 8 premature stop G1148ANBCCS F 12 2 bp insertion 2047insCT NBCCS S 12 1 bp insertion 2000insCNBCCS S 14 1 bp deletion 2583delC BCC S 5 premature stop CC1081TT BCC S15 14 bp deletion del 2704-2717

Discussion

[0334] These examples provide strong evidence that mutations of theNBCCS gene (PTC), the human homologue of Drosophila patched cause thenevoid basal cell carcinoma syndrome. Alterations predicted toinactivate the PTC gene product were found in six unrelated NBCCSpatients. Frameshift mutations were found in two sporadic patients butnot in their parents, and somatic mutations were identified in twosporadic tumors of the types seen in the syndrome. No known human tumorsuppressor has sequence similarity to PTC, and functionally PTC mayrepresent a novel type of neoplasia-related gene.

[0335] The Drosophila Patched Gene in Differentiation and Development

[0336] The patched gene is part of a signaling pathway that is conservedfrom flies to mammals. The Drosophila gene (ptc) encodes a transmembraneglycoprotein that plays a role in segment polarity (Hooper and Scott(1989) Cell 59: 751-765; Nakano et al. (1989) Nature 341: 508-513). Manyalleles of ptc produce an embryonic lethal phenotype with mirror-imageduplication of segment boundaries and deletion of the remainder of thesegments (Nusslein-Volhard et al. (1980) Nature 287: 795-801), buthypomnorphic alleles produce viable adults with overgrowth of theanterior compartment of the wing, loss of costal structures, and wingvein defects (Phillips et al. (1990) Development 110: 105-114). Geneticand functional studies have shown that one of the wild type functions ofptc is transcriptional repression of members of the Wnt and TGF-b genefamilies (Ingham et al. (1991) Curr. Opinion Genet. Develop. 5: 492-498;Capdevila et al. (1994) EMBO J. 13: 71-82. The mechanism of thisrepression is not known, and many other downstream targets of ptcactivity may exist.

[0337] The action of ptc is opposed by the action of members of thehedgehog gene family. Studies in Drosophila have demonstrated thathedgehog (hh) is a secreted glycoprotein which acts to transcriptionallyactivate both ptc-repressible genes and ptc itself (Tabata and Kombemc(1994) Cell, 76: 89-102; Basler and Struhl (1994) Nature, 368: 208-214).Thus, in a given cell type the activity of target genes results from abalance between hedgehog signaling, from adjacent cells and ptcactivation.

[0338] Mammalian Homologues of Patched

[0339] The human homologue (PTC) of Drosophilaptc, of this invention,displays no more than 67% identity at the nucleotide level and 61%identity at the amino acid level to the Drosophila gene. Thusidentification of a human homolog would have been difficult using eithera hybridization approach or by screening, an expression library withantibodies to the fly protein. The present date strongly suggest thatpatched is a single copy gene in mammals.

[0340] Analysis of fetal brain cDNA clones, and RACE experiments withepidermal RNA revealed the presence of two different 5′ ends for thehuman PTC gene (FIG. 3). The two human sequences diverge from the mousePTC CDNA (from 8 dpc embryo RNA) at the same position, and the mouseN-terminus more closely matches the Drosophila and C. elegans proteins.Analysis of the upstream genomic sequence of the C. elegans gene failedto reveal any homology to the two alternate human ends. These datasuggest that there are at least three different forms of the PTC proteinin mammalian cells; the ancestral form represented by the murinesequence, and the two human forms. The first in-frame methionine codonfor one of the human forms is in the 3rd exon, suggesting, that thisform of the mRNA either encodes an N-terminally truncated protein, oruses an alternate initiation codon. The second human form contains anopen reading, frame that extends through to the 5′ end, and may beinitiated by upstream sequences that have not yet been isolated. Theidentification of several potential forms of the PTC protein provides amechanism whereby a single PTC gene could feasibly play a role indifferent pathways. It will be important to determine the regulation ofthe different splice forms of Ptc mRNA as this may shed light on theapparent role of the gene in both embryonic development and growthcontrol of adult cells.

[0341] In adult humans PTC is expressed widely. Abundant transcript isfound in the kidney, liver, lung, brain, heart, skeletal muscle,pancreas, and skin. During murine development Ptc is expressed first at8.0 dpc in ventral neuroepithelial tissue in two separate domains alongthe midline. By day 9.5, transcripts are detected in the mesenchymesurrounding the neural tube as well. Expression is seen in thedeveloping somites in a rostral-caudal gradient. From day 10.0 to 12.5the transcript is present in the posterior ectoderm of each limb bud.Other sites of expression during this period include the inner surfacesof the pharyngeal arches, cells surrounding, the placodes of thevibrissae, and the genital eminence.

[0342] Several homologues of Drosophila hedgehog (hh) have beenidentified in vertebrates and, like the Drosophila gene, appear to beinvolved in pattern organization during development. The mostextensively studied of these is Sonic hedgehog (Shh). In the mouse,expression of Shh is normally detected in the notochord and theoverlying Doorplate region of the neural tube (Echelard et al. (1993)Cell 75: 1417-30). Apart from being, involved in midline signaling invertebrates, Shh is also expressed in a number of other tissues,including the developing, limbs, where it appears that Shh normallymediates the activity of the zone of polarizing activity (ZPA).

[0343] The expression of murine Ptc is found in a variety of tissuesknown to be responsive to Shh signaling. A detailed expression analysishas indicated that the pattern of expression closely follows changes inShh expression such that the transcripts are found mostly in adjacent,non-overlapping tissues. Ptc may be required for Shh signaling, andhhlptc interactions appear to have been conserved during evolution. Asin flies, Ptc transcription in mouse appears to be indicative of anadjacent hh signal. Accordingly, when interpreting the relationshipbetween known sites of Ptc expression and the NBCCS phenotype, it may beof value to consider the expression pattern of hedgehog gene familymembers since they have been characterized in much more detail invertebrates than ptc, especially in adult tissues.

[0344] While it is clear that genetically and functionally Ptc respondsto hh signaling, its structure does not make it an obvious hh receptor.Rather, it has been proposed that Ptc may be a transporter and thesubstrates are molecules which regulate the transcription of targetgenes.

[0345] The Role of PTC in Neoplasia

[0346] The data presented in this study strongly suggest the NBCCS genefunctions as a tumor suppressor. These examples show that germlinemutations underlying the NBCCS phenotype are inactivating, and thereforehereditary tumors have no functional copy of the gene. In addition,these examples provide the first direct evidence that sporadic basalcell carcinomas (BCCs) can arise with somatic loss of both copies of thegene. The role of PTC in other tumors related to the syndrome remains tobe explored.

[0347] That two known targets of ptc repression in Drosophila representgene families involved in cell-cell communication and cell signalingprovides a possible mechanism by which ptc could function as a tumorsuppressor. The ptc pathway has recently been implicated intumorigenesis by the cloning, of the pancreatic tumor suppressor gene,DPC4 (Hahn et al. (1996) Science 271: 350-353), which shows sequencesimilarity to Drosophila mad (mothers against dpp). The mad geneinteracts with dpp, a Drosophila TGF-b homologue specifically repressedby ptc.

[0348] The cell of origin of BCC has been greatly debated and currenttheory postulates a progenitor “stem cell” that is slow cycling, but ofgreat proliferative potential (Miller (1991) J. Am. Acad. Dermatol. 24:161-175). Through occasional cell division “transient amplifying cells”are formed, which further multiply before committing to terminaldifferentiation. The expression of patched and hedgehog gene familymembers in skin is not known. In Drosophila, ptc is found in membraneregions resembling cell adhesive junctions, and it colocalizes with PS2integrins, suggesting that the human homolog may normally play a role inepidermal differentiation based upon cell/cell interactions. The correctlocalisation of Drosophila ptc protein to these regions is alsodependent upon the interaction of the cells in a polarized epithelialsheet, an observation also supporting a role for ptc in cell adhesivestructures (Capdevila et al. (1994) Development 120: 987-998). Thepresent finding suggest that BCCs lack intracellular PTC signalingleading to an overexpression of the proliferative and cell/cellcommunication genes associated with hedgehog signaling. Alternatively,if PTC has a direct role in cell/cell interactions, proliferation mayresult from disruption at this level.

[0349] The Role of PTC in Developmental Anomalies

[0350] By analogy to embryonic expression of Ptc in the mouse, many ofthe features of NBCCS can be correlated with the presumed sites ofexpression of PTC in the developing human embryo (Table 4). For example,the skeletal anomalies involving the ribs, vertebrae and shoulders aremost likely due to disruption of PTC expression in the sclerotome. Inthe mouse embryo Ptc expression is detected in the ventral-medial cellsof the somites, a region which subsequently forms sclerotome (Hahn etal. (1996) supra.; Goodrich et al. (1996) Genes Dev. 10: 301-12). Inaddition, Shh has been implicated in the induction of sclerotome by longrange signaling from the notochord (Fan et al. (1995) Cell 81: 457-465).TABLE 4 Mouse Ptc expression and human NBCCS phenotype. Site ofexpression in mouse NBCCS Phenotype Pharyngeal arches Facialmalformations Jaw cysts (dental lamina derivative Neural tube Dysgenesisof the corpus callosum Eye anomalies Somites Spina bifida Vertebralfusion Rib anomalies Limb buds Short fourth metacarpals Polydactyly

[0351] The polydactyly observed in a subset of NBCCS patients is likelyto correlate with the expression of Ptc in the developing murine limb(Hahn et al. (1996) supra.; Goodrich et al. (1996) supra.). While theactual mechanisms remain unknown it seems clear that anterior posteriorpatterning of the limb is controlled by Shh signaling from the ZPA. Inthe early mouse limb bud Ptc expression correlates with Shh while atlater stages expression is detected in the periphery of the digitalcondensations in cells adjacent to those expressing Indian hedgehog(Ihh) (Goodrich et al. (1996) supra.) Therefore the polydactyly presentin NBCCS may result from perturbation of limb patterning due tomodulation of PTC. Similarly the occurrence of immobile thumbs in asmall percentage of NBCCS patients is consistent with an alteration towild type PTC function.

[0352] Craniofacial dysmorphology correlates with expression of Ptc inthe pharyngeal arches and derived structures. The jaw keratocysts anddental malformations, which are common features of NBCCS, are mostlikely explained by the observed expression of Ptc in the tooth bud andthe enamel knot (Vaahtokari et al. (1996) MOD, 54: 39-43; Goodrich etal. (1996) supra.). The pathogenesis of jaw cysts is almost certainlyrelated to the embryologic dental precursors. The epithelial lining ofkeratocysts is believed to arise from aberrant derivatives of the dentallamina, the precursor of tooth buds. The progenitor cells may havemigrated abnormally during development of the lamina or failed toinvolute at the appropriate stage of development.

[0353] The neurological components of NBCCS such as agenesis of thecorpus collosum, retinal colobomas, and possibly strabismus andmacrocephaly are consistent with the expression of Ptc in the developingbrain and neural system. Mental retardation, seen occasionally in thesyndrome, may be caused by contiguous gene deletions.

[0354] Under the classical two-hit model for the action of tumorsuppressors (Knudson (1971) Proc. Natl. Acad. Sci. USA, 68: 820-823) thefinding of developmental defects in a syndrome caused by hemizygousinactivation of this type of gene constitutes a paradox because loss ofjust one copy is thought to have little or no effect on cell function.It is possible that some of the discrete defects in NBCCS (e.g., spinabifida occulta, bifid ribs, and jaw cysts) can be explained by a two-hitmechanism. Like the neoplasms in cancer predisposition syndromes many ofthese defects are multiple and appear in a random pattern, but isolateddefects of the same type are seen occasionally in the generalpopulation. These anomalies might result from homozygous inactivation ofPTC in an early progenitor cell of the relevant tissue leading toabnormal migration or differentiation or perhaps failure to undergoprogrammed cell death. Allelic loss studies have in fact shown thatkeratocysts of the jaw are clonal abnormalities that arise withhomozygous inactivation of the NBCCS gene (Levanat et al. (1996) Nat.Genetics 12: 85-87). However, generalized or symmetric features such asovergrowth, macrocephaly, and facial dysmorphology almost certainly defythe two-hit paradigm and are probably due to haploinsufficiency. Itappears that many of the developmental defects seen in NBCCS patientsresult from perturbation of a dosage sensitive pathway during embryonicdevelopment. Based upon these observations one prediction would be thata heterozygous Ptc “knock-out” mouse would show relatively milddevelopmental anomalies and UV irradiation of the skin would result inmultiple basal cell carcinomas.

[0355] Phenotypic Variation in NBCCS

[0356] NBCCS is a disorder with almost 100% penetrance, but many of thefeatures show variable expression. An interesting correlate inDrosophila is that flies homozygous for non-lethal alleles show strikingvariability in expression of phenotypic features. Because these fliesare isogenic, the phenotypic differences cannot be explained bydifferent underlying, mutations or modifying genes (Phillips et al.(1990) supra.). Presumably the variability is a stochastic effect.

[0357] That there is more similarity in the human NBCCS phenotype withinfamilies than between families (Anderson et al. (1967) supra.) suggeststhat there may be some degree of genotype/phenotype correlation. Atpresent there are no clearly defined patterns of mutation distributionin NBCCS and all mutations found so far are predicted to causetruncation of the PTC protein. In families with BRCA1 terminationmutations, ovarian cancer is more common in patients with 5′ mutations,perhaps because mutant peptides may retain some wild-type function insome cell types but not in others (Gayther et al. (1995) Nature Genetics11: 428-433).

Example 3 Characterization of Ptc Germ Line Mutations in NBCCS Materialsand Methods

[0358] DNA Extraction

[0359] Odontogenic keratocyst tissue was placed in 200 μl STE buffer (50mM NaCl; 10 mM Tris-HCl pH8.0; 1 mM EDTA) containing 0.5% w/v SDS, 1μg/μl proteinase K and incubated at 37° C. for 24 hours. Followinginactivation of the proteinase K at 95° C. for 15 minutes, a sample of1-5 μl (approximately 25 ng DNA) was used directly for PCR.Constitutional DNA was obtained from peripheral blood lymphocytes orbuccal epithelial cells using standard DNA extraction procedures.Approximately 25 ng DNA was used directly for PCR.

[0360] Genbank Accession Numbers

[0361] DNA sequence data for the PTCH gene are available under accessionnumbers U43148 and U59464. The nucleotide numbering used in this Examplecorresponds to sequence U43148, whereas the amino acid residue numberingcorresponds to U59464.

[0362] PCR-SSCP Analysis of the PTCH Gene

[0363] Each of the 23 exons comprising the patched gene were amplifiedseparately using the primers and annealing conditions described in Hahnet al. (1996), supra. In brief, approximately 25 ng target DNA wasamplified in 30 μl 1×PCR reaction buffer containing 50 mM KCl; 10 mMTris-HCI (pH 9.0); 1.5 mM MgCl₂; 0.1% v/v Triton X-100; 200 μM eachdATP, dGTP, dTTP, 20 μM dCTP; 10 picomoles of each primer; 1.0μ Ci[α²P-dCTP] and 1.0 unit Taq. DNA polymerase (Promega, UK). Following 35cycles of amplification, 5 μl PCR product was added to 35 μl 10 mM EDTA,0.1% w/v SDS. 2 μl of this was added to 2 μl loading buffer containing95% v/v deionised formamide/20 mM EDTA/0.05% w/v bromophenol blue/0.05%w/v xylene cyanol, heated to 100° C. for 5 minutes, quenched on ice andloaded onto a 1×TBE/6% w/v non-denaturing polyacrylamide gel (5%C)containing (5%C) containing 5% v/v glycerol. Electrophoresis was at 350Vfor 18 hours. Gels were dried under vacuum and autoradiographed for 16hours at room temperature with intensifying screens.

[0364] Restriction SSCP PCR products obtained by amplification of exons14 and 17 were restriction enzyme digested with Alul and Hinfl,respectively, prior to SSCP analysis. An aliquot of 10 μl PCR productwas digested in a total volume of 20 μl 1×reaction buffer according tomanufacturers' instructions. 2 μl of restriction enzyme digested PCRproduct was mixed with 2 μl loading buffer and subjected to SSCPanalysis as described above.

[0365] DNA Sequencing of PTCH Exons

[0366] Exonic PCR products displaying altered mobilities by SSCPanalysis were purified using commercially available columns (Wizard PCRcolumns, Promega). PCR products were eluted in 20 μl TE and 2 μl usedfor DNA Thermosequenase (Amersham International plc) cycle sequencingaccording to manufacturer's instructions. Sequencing primers wereend-labelled with γ³²P-ATP (3000 Ci/mmol) using T4 polynucleotidekinase. DNA sequencing reactions were fractionated in 6% w/vpolyacrylamide/8M urea/1×TBE gels for 2 hours at 2000V. Gels were driedunder vacuum and autoradiographed for 8 hours at room temperature withintensifying screens.

Results

[0367] Keratocyst DNA from a total of 16 NBCCS patients was screened bySSCP-PCR. 10 single exonic PCR products displaying alteredelectrophoretic mobilities were detected and analyzed further by DNAsequence analysis. In addition, variant bands in multiple samples (i.e.indicative of common polymorphisms) were also seen in PCR productsencompassing 4 exons.

[0368] Four mutations were identified following direct DNA sequencing ofPCR amplified exons displaying SSCP variant bands. Mutations could notbe detected in the remaining 6 variant PCR products. Therefore, all 23exons from the samples in which a mutation had not been identifiedinitially were amplified and sequence in their entirety. However, onlyone additional mutation was detected by this method.

[0369] Exon 5 693 insC

[0370] A single cytosine residue insertion at position 693 was detectedin a 42 year old male NBCCS patient. This introduces a frameshiftmutation by the creation of a premature stop codon at amino acid residue252. This mutation also creates a BstNI restriction enzyme site. DNAfrom the patient and 4 unaffected family members was amplified andrestriction enzyme digested with BstNI. As predicted, only the productfrom the NBCCS patient was cut by the restriction enzyme.

[0371] Exon 17 2988 del8bp

[0372] Following Hinfl restriction enzyme digestion of exon 17 PCRproducts and SSCP analysis, 2 variant bands were seen. Direct DNA cyclesequencing of these amplicons revealed 2 mutations. An 8 bp deletion wasdetected in DNA from a 12 year old male NBCCS patient. This frameshiftmutation introduces a stop codon at amino acid residue 1141. The patienthas macroephaly, hypertelorism, supra-orbital ridges, prognathism,plantar but not palmar pitting and an accessory nipple. In addition, atage 10 years, the patient had undergone surgical removal of 3 maxiallaryand mandibular odontogenic keratocysts.

[0373] Exon 173014 insA

[0374] A adenosine insertion at base 3014 was detected in an 18 year oldfemale NBCCS patient. This results in a frameshift mutation (tyrosine toSTOP) codon at amino acid residue 1009. This patient was frontalbossing, hypertelorism, falx calcification, bifid 3rd, 4th, 5th and 6thribs and has undergone enucleation of 5 maxillary and mandibularodontogenic keratocysts.

[0375] Exon 21 3538 deIG

[0376] A guanosine base deletion at residue 3538 was identified in a 38year old female NBCCS patient. This frameshift mutation introduces astop codon at amino acid residue 1190. The causal nature of the mutationwas confirmed by analysis of DNA from the proband's father, from whomshe has inherited the disorder. Direct DNA sequencing of exon 21 fromthe father also revealed a guanosine base deletion at residue 3538.

[0377] Exon 22 G4302T

[0378] Direct DNA sequencing of 23 exons from a 30 year old female NBCCSpatient revealed a G-T substitution at nucleotide 4302. This cases aglutamic acid to aspartic acid (E-D) substitution at amino acid residue1438. The patient has been confirmed as a case of NBCCS and hasundergone removal of 11 basal cell carcinomas.

[0379] PTCH Gene Polymorphisns

[0380] Using the SSCP conditions described, the inventors observedpolymorphisms in PCR products amplified from exons 6, 11, 14 and 15. NoDNA sequence alterations were detected following analysis of exonicsequences. Therefore, it is likely that these represent intronic DNAsequence polymorphisms. Also, an exonic DNA sequence polymorphism(C306T) was disclosed in exon 2. This base substitution was observed inDNA from subject LDI-1, in which a “causative” 3538delG mutation hadalready been identified, as well as other unrelated NBCCS patients.

[0381] In this example, the inventors identified 5 novel, germ linemutations from patients with the NBCC cell, consistent with the role ofpatched as a human tumour suppressor gene. Four mutations causeframe-shift or none-sense mutations resulting in a truncated PTCHprotein; the fifth mutation is a glutamic acid to aspartic acidsubstitution close to the 3′-carboxyl terminus of the PTCH protein. Thiswas the only base change detected following direct DNA sequencing of all23 PTCH exons from patient #5. Although this represents a conservativeamino acid substitution and, therefore, may be a polymorphism and not amutation, this glutamic acid residue is conserved between human, mouseand chicken PTCH proteins and is likely to be functionally important.

Example 4 Mutations of the Ptc Gene in NBCCS Define Clinical Phenotype

[0382] Example 3 defined mutation in individuals with NBCC syndrome. Inthis Example, further mutations are identified.

Materials and Methods

[0383] The patients were diagnosed according to the clinical criteria ofShanley et al (1994). Seventy NBCCS patients were fully analyzed bysingle strand conformation polymorphism (SSCP) and heteroduplex analysisas previously described (Hahn et al., supra.) with primers for allcoding exons except for exon 1b (alternative first exon). Primersequences were as hereinbefore described as well as Hahn et al (1996)with the exception of exons 12, 12b and 20. Exon 12b is an additionalexon resulting from the discovery that exon 12 (Hahn et al. 1996)consists of two distinct exons. The PTC gene, therefore, consists of 23coding exons. Where possible, DNA was also analyzed from the parents ofcases in which PTC mutations were found in order to determine at amolecular level whether the mutation was sporadic or familial. Anysamples showing SSCP variants were sequenced as previously described(Wicking et al. (1997) Am. J. Hum. Genet. 60: 21-26).

[0384] Paternity testing was carried out using microsatellite markers inthe parents of the eight sporadic cases using fluorescent primers andGenescan.

Results

[0385] PTCH mutations were identified in a total of 32 NBCCS cases.Twenty-eight of these are described in Example 3. This Example presentsfour novel mutations (Table 5). Of these, three are frameshift mutationsand one is a putative splice variant. The inventors have, therefore,detected PTCH mutations in 32/70 (46%) NBCCS cases by analysis of allexons except exon 1b. The majority of these (27/32; 84%) are proteinterminating mutations. In addition, eight sporadic cases were identifiedby the absence of the relevant disease-associated mutation in eitherparent (Table 6). Paternity testing was performed in these eight cases,using four microsatellite markers and in none was any inconsistencyidentified.

[0386] This Example presents eight individuals with NBCCS whom theinventors have shown to carry mutations in the PTCH gene. In all cases,the parent did not carry the disease-related mutation, and non-paternitywas shown to be unlikely by analysis of highly informativemicrosatellite markers. In addition, the inventors report four NBCCSindividuals with germline PTCH mutations. Three out of four of thesewould result in premature protein truncation.

[0387] The ability to confirm the diagnostic status of relatives ofNBCCS cause by DNA analysis allows further definition of the clinicaland radiological criteria used to diagnose NBCCS. Of the eight NBCCSindividuals shown to have new mutations in the PTCH gene in this studyonly two (JRN250, DD25) clearly represented sporadic cases based onclinical and radiological examination of both parents. In one case(JHK551) neither parent had been examined. In all other cases one parentshowed at least one feature associated with NBCCS such as multiple BCCS,a high arched palate or macroephaly. TABLE 5 PTCH MUTATIONS IN NBCCSINDIVIDUALS Patient Exon Mutation Effect on coding JK211 12 1711insCFrameshift, truncation MB229 12 1639insA Frameshift, truncation CW424 162707delC Frameshift, truncation NB88 Intron 17 3157-2A-> G Putativesplice variant

[0388] TABLE 6 MUTATIONS OF PTCH IN NBCCS PATIENTS Patient MutationEffect Parental phenotype DD25 C2050T Nonsense Parents both clinicallyand radiologically negative but multiple BCCs in history of grandmother.JHG547 C391T Nonsense Father has had > 10 BCCs from 6th decade butradiologically negative. Mother clinically and radiologically negative.BK273 244delCT Frameshift Father has high arched palate, macrocephalyand dense falcine calcification (age 53) but below maximum biparietaldiameter. The Mother clinically and radiologically negative. JHK551271insA Frameshift Negative family history but neither parent examined.JRN250 929delC Frameshift Both parents clinically and radiologicallynegative. MP264 2183delTC Frameshift Father has high arched palate,macrocephaly and three “pits” on soles but radiologically negative.Mother clinically and radiologically negative. KS356 2583delC FrameshiftFather has had three unconfirmed BCCs (age 65) but radiologicallynegative. Mother negative clinically. No radiological examinations.JK211 1711insC Frameshift Father has high arched palate and has hadabout 20 BCCs (from age 70) but also multiple solar keratoses,keratoacanthoma and squmous cell carcinomas. He is radiologicallynegative. Mother clinically and radiologically negative.

Example 5 Medulloblastomas of the Desmoplastic Variant Carry Mutation ofthe Human Ptc Gene

[0389] In this Example, the inventors detected non-conservative PTCmutations in three of 11 sporadic cases of desmoplastic medulloblastomas(Mbs) but none in 57 tumours with classical (non-desmoplastic)histology. In two of the tumours with mutations and in two additionaldesmoplastic cases, LOH was found at 9q22. These findings suggest thatPTC represents a tumour suppressor gene involved in the development ofthe desmoplastic variant of MB.

Materials and Methods

[0390] Patient and Tumours, Cell Lines.

[0391] A total of 68 medulloblastoma samples were analyzed, 64 sampleswere obtained from MB tumors and 4 from the previously describedmedulloblastoma cell lines D283Med, D341Med, Daoy, and MHH-MED-1(Pietsch et al. (1994) Cancer Res. 54: 3278-3287). In two patients, theinventors were able to study both the primary and the recurrent tumors.Constitutional DNA from peripheral blood-was available in 40 patients.DNA samples from peripheral blood from healthy Caucasian volunteers wereused as controls. A sample of normal cerebellum was analyzed. Thisbiopsy specimen was from an adult patient with a cerebellar vascularmalformation and was found to be normal upon histopathological review.The patients' age ranged from 1 month to 59 years; there were 46 malesand 20 females. None of the patients had clinical signs of NBCCS or hadfirst degree relatives with NBCCS. All tumors were diagnosed accordingto the revised WHO classification of brain tumors using standardhistological methods including HE and reticulin stains andimmunohistochemical reactions (Kleihues et al. (1993) Histologicaltyping of tumours of the central nervous system, Springer Verlag, NewYork). Differentiation was assessed by immunostaining for embryonalneural cell adhesion molecule (NCAM), neuron-specific enolase,synaptophysin and glial acidic fibrillary protein. Frozen tumour sampleswere obtained at the time of surgical resection, snap frozen in liquidnitrogen and stored at −80° C.

[0392] DNA Extraction, LOH Analysis.

[0393] Tumour fragments were selected for extraction of DNA aftercareful examination of corresponding frozen sections to excludecontaminating necrotic debris or normal cerebellar tissue and todetermine the histological characteristics of the tumors. DNA wasextracted by standard proteinase K digestion and phenol/chloroformextraction (Albrecht et al, 1994). Loss of heterozygosity was determinedby microsatellite analysis with the markers D9S287 and D9S197 which weretightly linked to the PTC gene and with two additional markers on 9q(D9S302, D9S303) essentially as previously described (Albrecht et al.(1994) Neuropathol. Appl. Neurobiol. 20:74-81; Kraus et al. (1996) Int.J. Cancer 67: 11-15).

[0394] SSCP Analysis and DNA Sequencing

[0395] SSCP analysis of exons 2-22 was performed using 22 primer pairs(previous Examples and Hakin et al, 1996). PCR containing 50 mM KCl,1.0-2.5 mM MgCl₂10 mM Tris-HCl (pH 8.5), 0.01% w/v gelatin and 200 mM ofeach dNTP, 2 μM of the primers and 0.25 units Taq polymerase (Gibco-BRL)on a Uno Thermoblock cycler (Biometra). The products were analyzed onpolyacrylamide gels with different acrylamide concentrations andacrylamideibisacrylamide ratios. Gel composition and electrophoresisconditions were optimized for each individual primer pair. The singleand double strands were visualized by silver staining as previouslydescribed (Albrecht et al, 1994). PCR products which showed a gelmobility shift were excised from the wet gel, eluted (Koch et al, 1996)and reamplified by PCR with the same primers. The resulting productswere purified using spin columns (Qiagen quick spin), and 20 ng used tocycle sequencing with a fluorescent dideoxy terminator kit (ABI). Theproducts were analyzed on an Applied Biosystems model 373A DNAsequencer.

[0396] Isolation of RNA, Quantitative RT-PCR for PTCH mRNA.

[0397] Total cellular RNA was extracted by lysis in guanidiniumisothiocyanate and ultracentrifugation through a cesium chloride cushion(Koch et al, 1996) or by extraction with the Trizol™ reagent (Gibco-BRL)following the manufacturer's instructions. Again, individual sampleswere preexamined by frozen section histology to document thehistopathological appearance of the specimen. Contaminating residualgenomic DNA was removed by digestion with RNAse free DNAse (Boehringer).RNA standards with internal deletions for human PTC and the housekeepinggenes β₂ -microglobulin and GAPDH were generated by in vitro mutagenesisand in vitro transcription (Horton and Pease (1991) in DirectMutagenesis—Practical Approach, McPerson, ed., pp. 217-247 (IRL Press,Oxford). In order to achieve a semi-quantitative assessment,pre-evaluated amounts of the specific standards RNAs covering theequimolar range of the corresponding mRNA transcripts were added to theMB sample RNAs which were then reverse transcribed using theSuperScript™ Preamplification System (Gibco-BRL) with random hexamers asprimers in a final volume of 10 μl. 0.5 μl of the cDNA was used as atemplate in RT-PCR reactions for amplification of PTCH, and thehousekeeping genes. The primers used were: PTCH,5′ACATGTACAACAGGCAGTGG-3 [SEQ ID NO: 61] and 5′-GCAAGGAGGTTTACCTAGG-3′[SEQ ID NO.62], product size, wild type 192 bp, standard 182 bp; GAPDH,5′-TGCCAAGGCTGTGGGCAAGG-3′ [SEQ ID NO: 63] and5′-GCTTCACCACCTTCTTGATG-3′ [SEQ ID NO: 64] product size, wild type 152bp, standard 142 bp; β₂-microglobulin, 5′-GCTGTGACAAAGTCACATGG-3′ [SEQID NO: 65] and 5′-GATGCTGCTTACATGTCTCG-3′ [SEQ ID NO: 66], product size,wild type 148 bp, standard 130 bp. One of the primers for each gene waslabeled with a fluorescent dye. All primers were chosen from adjacentexons spanning intronic sequences in order to avoid signals of the cDNAproduct size caused by residual genomic DNA. The PCR products wereseparated and analyzed on an Applied Biosystems model 373A DNA sequencerusing the Genescan software (ABI). The expression levels of theindividual genes were calculated from the signal ratios of the samplesto the standards. The relative expression of PTCH mRNA to thehousekeeping genes was defined as the ratio of the respective expressionlevels (FIG. 10).

[0398] The human PTCH gene spans 34 kB and has at least 23 exons. SSCPscreening of DNA samples from 68 sporadic MBs revealed band shifts in 6samples (Table 7). Three of these were identified as silentpolymorphisms. In three other tumors, the variants were not found in thecorresponding germline DNA or in normal control DNA samples. Twomutations in exons 6 and 10, respectively, resulted in a frame shiftwith premature truncation of the protein (Table 7 and FIG. 9). The thirdmutation (D86) was a six base pair in frame deletion in exon 10 leadingto the deletion of two amino acids in transmembrane region 3. Thisdeletion may cause significant structural alterations of the PTCHprotein and may result in loss of function. This was the case in tumoursD86 and D322 which showed LOH as well as mutated PTC allele. In caseD292 without detectable LOH at 9q, only a single SSCP band shift wasfound (in exon 10). Mutations of the other allele may be present but maynot have been detected by SSCP screening because of the limitedsensitivity of SSCP. Only the coding exons were screened so thatmutations in other regions such as regulatory domains would not havebeen identified with this approach. A systematic sequencing analysis mayuncover additional PTCH mutations in Mbs.

[0399] In this Example, mutations were detected in a distincthistopathological variant or medulloblastomas (MB), the so-calledmodular or “desmoplastic” MB. According to the WHO classification thisvariant is characterized by islands of lower cellularity surrounded bydensely packed, highly proliferative cells which produce a denseintercellular reticulin fiber network. The more frequent “classical” MBlacks this nodular appearance and reticulin pattern. TABLE 7 MUTATIONALANALYSIS of the PTCH GENE IN MEDULLOBLASTOMAS Tumour MB variant Age/sexLOH on 9q Exon Nucleotide change Protein change a, Mutations D 86desmoplastic 4y, male yes 10 1444de16 del Gly-Leu D 292 desmoplastic 1y,female no 10 1393insTGCC frameshift, truncation D 322 desmoplastic 51y,male yes 6 887deIG frameshift, truncation b, Polymorphisms D 230 11classical 13y, female no 13 C2037T no D 338 classical 13y, male n.a. 2C306T no D 358 classical 10y, female na. 2 C306T no

[0400] TABLE 8 EXPRESSION OF THE PTCH GENE IN MEDULLOBLASTOMAS PTCH mRNAmutation mRNA expression ratio MB detected by expression ratio PTCH/β2-Sample subtype LOH on 9q SSCP PTCH/GAPDH microglobulin D 338 classicaln.a.* no  5.8 (3.9-7.4)** 10.3 (8.2-14.2) D 230 II classical no no  1.7n.a. D 286 classical n.a. no  1.2 n.a. D 245 II classical no no  0.7n.a. D 446 classical no no  1.8 (0.8-2.8)  1.5 (1.0-1.8) D 447 classicalno no  0.6 n.a. D 86 desmopl. yes yes, exon 10  3.6 (1.4-5.4)  2.6(1.7-3.9) D 292 desmopl. no yes, exon 10  0.6 (0.6-0.6)  0.3 (0.3-0.4) D322 desmopl. yes yes, exon 6  1.1 n.a. D 448 desmopl. yes no  4.3(3.3-5.5)  2.9 (2.5-3.6) D 398 desmopl. no no 26.5 (22.4-30.0) 18.4(12.0-24.4) D 444 desmopl. yes no  4.1 (3.7-4.7)  4.5 (3.3-5.5) D 365***desmopl. n.a. no  0.3 (0.2-0.4)  0.1 (0.1-0.2) Cerebellum — n.a. n.a. 2.5 (1.55-3.64) 1.76 (1.42-2.09)

Example 6

[0401] Most Germ Line Mutations in the NBCCS Gene Lead to a PrematureTermination of the PATCHED Protein

[0402] In this Example, the inventors screened DNA samples from 71unrelated NBCCS individuals for mutations in the PTCH exons using singlestrand conformational polymorphism (SSCP) analysis. In total, 28mutations were identified and characterised by direct sequencing of PCRproducts. The majority of these mutations (86%) lead to prematuretruncation of the PTCH protein. Analysis of phenotype in individualswith truncating mutations revealed no statistically significantcorrelation between genotype and phenotype in NBCCS.

Materials and Methods

[0403] Subjects and Samples

[0404] The patients, most of whom were from Australia and New Zealand,were diagnosed according to the clinical criteria in Shanley et al.(1994) supra. Of the 71 NBCCS patients analyzed 25 show clear familialpresentation and 46 are apparently sporadic.

[0405] SSCP Analysis

[0406] A combined SSCP and heteroduplex analysis was performed aspreviously described (Hahn et al. (1996) supra). DNA from 71 unrelatedNBCCS individuals was amplified with primers to all but exons 1b(alternative first exon homologous to murine exon 1), 12 and 20 (forwhich acceptable primers are not yet available) of the human PTCH gene.Primer sequences and conditions were as hereinbefore described as wellas (Hahn et al. (1996) supra.).

[0407] Sequencing

[0408] DNA from samples showing SSCP variants was reamplified andpurified for automated sequencing using PCR Spinclean Columns (ProgenIndustries). DNA concentration was ascertained by agarose gelelectrophoresis and 25-35 ng of product was used for each automatedsequencing run. Cycle sequencing was performed using Amplitaq FSpolymerase and dye labelled terminator chemistry (Perkin Elmer Cetus),and samples were analyzed on an PEC 373A electrophoresis apparatus.Where mutations were confirmed by manual sequencing the purifiedproducts were ligated into GEM-T vector (Promega) and the resultingclones were sequenced with a T7 Sequencing Kit (Pharnacia).

[0409] Southern Analysis

[0410] Southern blots were made as previously described (Chenevix-Trenchet al. 1992), using EcoRI and HindIII restriction enzymes and hybridizedwith PTCH cDNA probes 13 B and 16C.

[0411] Statistical Analysis

[0412] Linear regression analysis was used to examine genotype-phenotypeassociations.

Results

[0413] Identification of PTCH Mutations

[0414] DNA from 71 individuals with NBCCS was screened for mutations inthe PTC gene by a combined SSCP/heteroduplex analysis. Based uponsequencing of samples showing SSCP variation, 28 putativedisease-associated mutations have been fully characterised (Table 9).While one mutation, a CT deletion at nucleotide position 244, was seenin 3 apparently unrelated individuals, all other mutations were onlydetected in a single NBCCS family. No clustering of mutations has beenobserved, with mutations identified in most exons and in positionscorresponding to all of the major domain types of the PTC protein (FIG.11).

[0415] The majority (24/28; 86%) of mutations detected are predicted toresult in truncation of the PTCH protein either by introduction of astop codon or by frameshift due to insertion or deletion (Table 9). Twoof the mutations were predicted to be splice variants based on the factthat one altered a consensus 3′ splice site, while the other involved aninsertion of 21 bp in intron 10, 8 bp upstream of the start of exon 11.This second mutation was presumed to cause aberrant splicing based onthe fact that it moves the branch site from position 27 to 48 bpupstream of the 3′ splice site. This 7 bp consensus sequence isgenerally located approximately 18 to 37 bp upstream of the splice site,and its location is considered to be important in the splicing reaction.The remaining two mutations are missense mutations which both alterresidues within transmembrane domains of the PTCH protein. One (PP) is atransversion of GAT to TAT at nucleotide 1525, substituting a tyrosinefor an aspartate in the fourth transmembrane domain; the other (RS) is atransversion of a GGC to CGC at nucleotide 3193, substituting anarginine for a glycine in the ninth transmembrane domain of the PTCHprotein.

[0416] In addition to disease-associated mutations, several variantswere designated polymorphisms based on their presence in unaffectedindividuals, or the finding that the underlying sequence changes did notalter the encoded amino acids. For samples in which no SSCP variationwas found, the sequence of each exon of the PTC gene is currently beingdetermined. DNA from 38 patients in which a mutation was not found bySSCP was also examined by Southern analysis using probes which span thegene. Variants were detected in two patients. In each case, a singleadditional band (3 and 3.75 kb respectively) was seen on HindIII blots.No variants were present in these individuals on EcoRI, BamHI and Pst Idigests, therefore, those seen on HindIII blots are unlikely torepresent gross rearrangements. The probability of at least one of thesevariants being a disease-related point mutation is increased by itssegregation with disease in a family. No family members were availablefor study in the case of the second variant. The underlying mutations inthese individuals are yet to be determined.

[0417] Analysis of Genotype-phenotype Associations

[0418] Although most of the mutations found to date are predicted totruncate the protein, it remains to be determined whether all ablate itsfunction. In order to address this, preliminary analysis ofgenotype-phenotype associations in this complex syndrome was performed,based on the 24 families with protein-truncating mutations. Severalaspects of the NBCCS phenotype were used as approximate parameters ofdisease severity. The inventors examined the number of major features(BCCs, jaw cysts, pitting and falcine calcification) seen in individualsat the time of diagnosis, the age at which the individual manifestedBCCs and the age at which jaw cysts were detected. Individuals under theage of 20 years were not included in this analysis due to theage-dependent expression of these features. Similarly, analysis of ageof BCC onset was restricted to Australasian patients to limit theinfluence of ultraviolet exposure in promoting BCC development. When amutation was known to be present in a number of individuals within afamily, phenotypic data were averaged across all relevant familymembers. No correlations between the age of onset of BCCs (R²=0.001) orjaw cysts (R²=0.023), or the number of major features (R²=0.015), andnucleotide position of the mutation, were found, indicating that forthese features at least, there is no clear correlation between phenotypeand location of the truncating mutation. Although it was not appropriateto use statistical analyses to compare truncating mutations withmissense and splice variants due to the small number of the latter typeof mutations, the individuals the inventors have analyzed with missenseand splice mutations show a classic NBCCS phenotype and would not beclassed as mildly affected.

[0419] The phenotypes of individuals in the three families which share acommon mutation (244delCT) were evaluated, and shown to varyconsiderably. All five affected members of family CB have a cleft orvery high arched palate but this was not observed in the HC or BKfamilies, both of whom show the typical range of NBCCS features. Thissuggests that the molecular nature of the PTC mutation is not entirelyresponsible for the phenotype in NBCCS. Interestingly, BK carries a newmutation of PTC so this mutation must have arisen at least twice.

[0420] The inventors have identified mutations in the PTC gene in 28unrelated individuals with NBCCS and found no evidence of anyassociation between genotype and phenotype. In 24 families withprotein-truncating mutations, no significant correlation betweenphenotype and location of the truncating mutation was found. Thisdiffers from breast and ovarian cancer where 3′ mutations in the BRCA1gene are less likely to predispose to ovarian cancer than are 5′mutations presumably because of residual activity of proteins resultingfrom 3′ mutations. TABLE 9 GERM-LINE MUTATIONS IN THE PTC GENE IN NBCCSINDIVIDUALS EFFECT ON PATIENT^(a) EXON MUTATION^(b) CODING^(c) MissensePP (S) 11 G1525T D-Y at 513 RS (S) 18 G3193C G-R at 1069 JHG (S)  3C391T R-X at 135 JM (F)  8 G1148A^(d) W-X at 387 TM (S) 10 G1368A W-X at460 DD (S) 13 C2050T Q-X at 688 BH (S) 13 C2068T Q-X at 694 PB (F) 17C3015a Y-X at 1009 Insertions, Deletions, and Duplications HC, CB(F);BK(S)^(c)  2 244delCT Frameshift JHK (S)  2 271insA Frameshift GS (S)  3464insAC Frameshift MC (F)  6 804del37^(d) Frameshift JRN (S)  6 929delCFrameshift DS (F) 10 1370del76 Frameshift CM (S) 11 1497dup8 FrameshiftMP (F) 13 2183delTC Frameshift TH (F) 14 2320insA Frameshift LK (S) 142392delA Frameshift DC (S) 15 2574delA Frameshift KS (S)^(c) 152583delC^(d) Frameshift WS (F) 15 2596complex^(f) Frameshift DE (F) 162748insC Frameshift JRD (F) 16 2749dup7 Frameshift JW (S) 19 3352delATFrameshift Splicing AE (F) Intron 7 A1055-2C 3′ splice site IMc (S)Intron 10 1493-8ins2l Putative splice variant Polymorphisms^(g)  2 A orT at 312 No change I108  3 T or C at 417 No change T143  4 A or G at 588No change E200  5 A or G at 723 No change T245  7 T or C at 1023 Nochange G345 Intron 10 G or C at 1493 − 39 Intron 11 A or T at 1591 + 29Intron 18 delTT at 3294 + 27 22 T or C at 3933 No change L1315

[0421] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference for all purposes.

0 SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES:84 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 6568 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1..6568 (D) OTHER INFORMATION: /note= “humannevoid basal cell carcinoma syndrome (NBCCS) (PATCHED (PTC)) cDNA” (ix)FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 442..4332 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 1: GAAGGCGAGC ACCCAGACGG GGGCCCGCCG GGGTCGCGGCCAGCGCCGGG GAAATGCCGC 60 GCCGGGGAGC AGCATGCGCC GGCCTGAGCC CTTCCCTTTGCACTCGGCTG TTTTTTACGT 120 TTAACCAGAA AGGAAGGGAG AGGAGGGAAA GATCCATGTGGCTGCCCTCT TCCGATCACA 180 AATATTGTCG GGAAGGCTAC TGGCCGGAAA GCGCCGCTGTGGCTGAGAGC GAAGTTTCAG 240 AGACTCTTAT TTAAACTGGG TTGTTACATT CAAAAAAACTGCGGCAAGTT CTTGGTTGTG 300 GGCCTCCTCA TATTTGGGGC CTTCGCGGTG GGATTAAAAGCAGCGAACCT CGAGACCAAC 360 GTGGAGGAGC TGTGGGTGGA AGTTGGAGGA CGAGTAAGTCGTGAATTAAA TTATACTCGC 420 CAGAAGATTG GAGAAGAGGC TATGTTTAAT CCTCAACTCATGATACAGAC CCCTAAAGAA 480 GAAGGTGCTA ATGTCCTGAC CACAGAAGCG CTCCTACAACACCTGGACTC GGCACTCCAG 540 GCCAGCCGTG TCCATGTATA CATGTACAAC AGGCAGTGGAAATTGGAACA TTTGTGTTAC 600 AAATCAGGAG AGCTTATCAC AGAAACAGGT TACATGGATCAGATAATAGA ATATCTTTAC 660 CCTTGTTTGA TTATTACACC TTTGGACTGC TTCTGGGAAGGGGCGAAATT ACAGTCTGGG 720 ACAGCATACC TCCTAGGTAA ACCTCCTTTG CGGTGGACAAACTTCGACCC TTTGGAATTC 780 CTGGAAGAGT TAAAGAAAAT AAACTATCAA GTGGACAGCTGGGAGGAAAT GCTGAATAAG 840 GCTGAGGTTG GTCATGGTTA CATGGACCGC CCCTGCCTCAATCCGGCCGA TCCAGACTGC 900 CCCGCCACAG CCCCCAACAA AAATTCAACC AAACCTCTTGATATGGCCCT TGTTTTGAAT 960 GGTGGATGTC ATGGCTTATC CAGAAAGTAT ATGCACTGGCAGGAGGAGTT GATTGTGGGT 1020 GGCACAGTCA AGAACAGCAC TGGAAAACTC GTCAGCGCCCATGCCCTGCA GACCATGTTC 1080 CAGTTAATGA CTCCCAAGCA AATGTACGAG CACTTCAAGGGGTACGAGTA TGTCTCACAC 1140 ATCAACTGGA ACGAGGACAA AGCGGCAGCC ATCCTGGAGGCCTGGCAGAG GACATATGTG 1200 GAGGTGGTTC ATCAGAGTGT CGCACAGAAC TCCACTCAAAAGGTGCTTTC CTTCACCACC 1260 ACGACCCTGG ACGACATCCT GAAATCCTTC TCTGACGTCAGTGTCATCCG CGTGGCCAGC 1320 GGCTACTTAC TCATGCTCGC CTATGCCTGT CTAACCATGCTGCGCTGGGA CTGCTCCAAG 1380 TCCCAGGGTG CCGTGGGGCT GGCTGGCGTC CTGCTGGTTGCACTGTCAGT GGCTGCAGGA 1440 CTGGGCCTGT GCTCATTGAT CGGAATTTCC TTTAACGCTGCAACAACTCA GGTTTTGCCA 1500 TTTCTCGCTC TTGGTGTTGG TGTGGATGAT GTTTTTCTTCTGGCCCACGC CTTCAGTGAA 1560 ACAGGACAGA ATAAAAGAAT CCCTTTTGAG GACAGGACCGGGGAGTGCCT GAAGCGCACA 1620 GGAGCCAGCG TGGCCCTCAC GTCCATCAGC AATGTCACAGCCTTCTTCAT GGCCGCGTTA 1680 ATCCCAATTC CCGCTCTGCG GGCGTTCTCC CTCCAGGCAGCGGTAGTAGT GGTGTTCAAT 1740 TTTGCCATGG TTCTGCTCAT TTTTCCTGCA ATTCTCAGCATGGATTTATA TCGACGCGAG 1800 GACAGGAGAC TGGATATTTT CTGCTGTTTT ACAAGCCCCTGCGTCAGCAG AGTGATTCAG 1860 GTTGAACCTC AGGCCTACAC CGACACACAC GACAATACCCGCTACAGCCC CCCACCTCCC 1920 TACAGCAGCC ACAGCTTTGC CCATGAAACG CAGATTACCATGCAGTCCAC TGTCCAGCTC 1980 CGCACGGAGT ACGACCCCCA CACGCACGTG TACTACACCACCGCTGAGCC GCGCTCCGAG 2040 ATCTCTGTGC AGCCCGTCAC CGTGACACAG GACACCCTCAGCTGCCAGAG CCCAGAGACC 2100 ACCAGCTCCA CAAGGGACCT GCTCTCCCAG TTCTCCGACTCCAGCCTCCA CTGCCTCGAG 2160 CCCCCCTGTA CGAAGTGGAC ACTCTCATCT TTTGCTGAGAAGCACTATGC TCCTTTCCTC 2220 TTGAAACCAA AAGCCAAGGT AGTGGTGATC TTCCTTTTTCTGGGCTTGCT GGGGGTCAGC 2280 CTTTATGGCA CCACCCGAGT GAGAGACGGG CTGGACCTTACGGACATTGT ACCTCGGGAA 2340 ACCAGAGAAT ATGACTTTAT TGCTGCACAA TTCAAATACTTTTCTTTCTA CAACATGTAT 2400 ATAGTCACCC AGAAAGCAGA CTACCCGAAT ATCCAGCACTTACTTTACGA CCTACACACC 2460 AGTTTCAGTA ACGTGAAGTA TGTCATGTTG GAAGAAAACAAACAGCTTCC CAAAATGTGG 2520 CTGCACTACT TCAGAGACTG GCTTCAGGGA CTTCAGGATGCATTTGACAG TGACTGGGAA 2580 ACCGGGAAAA TCATGCCAAA CAATTACAAG AATGGATCAGACGATGGAGT CCTTGCCTAC 2640 AAACTCCTGG TGCAAACCGG CAGCCGCGAT AAGCCCATCGACATCAGCCA GTTGACTAAA 2700 CAGCGTCTGG TGGATGCAGA TGGCATCATT AATCCCAGCGCTTTCTACAT CTACCTGACG 2760 GCTTGGGTCA GCAACGACCC CGTCGCGTAT GCTGCCTCCCAGGCCAACAT CCGGCCACAC 2820 CGACCAGAAT GGGTCCACGA CAAAGCCGAC TACATGCCTGAAACAAGGCT GAGAATCCCG 2880 GCAGCAGAGC CCATCGAGTA TGCCCAGTTC CCTTTCTACCTCAACGGCTT GCGGGACACC 2940 TCAGACTTTG TGGAGGCAAT TGAAAAAGTA AGGACCATCTGCAGCAACTA TACGAGCCTG 3000 GGGCTGTCCA GTTACCCCAA CGGCTACCCC TTCCTCTTCTGGGAGCAGTA CATCGGCCTC 3060 CGCCACTGGC TGCTGCTGTT CATCAGCGTG GTGTTGGCCTGCACATTCCT CGTGTGCGCT 3120 GTCTTCCTTC TGAACCCCTG GACGGCCGGG ATCATTGTGATGGTCCTGGC GCTGATGATC 3180 GTCGAGCTGT TCGGCATGAT GGGCCTCATC GGAATCAAGCTCAGTGCCGT GCCCGTGGTC 3240 ATCCTGATCG CTTCTGTTGG CATAGGAGTG GAGTTCACCGTTCACGTTGC TTTGGCCTTT 3300 CTGACGGCCA TCAGCGACAA GAACCGCAGG GCTGTGCTTGCCCTGGAGCA CATGTTTGCA 3360 CCCGTCCTGG ATGGCGCCGT GTCCACTCTG CTGGGAGTGCTGATGCTGGC GGGATCTGTC 3420 TTCGACTTCA TTGTCAGGTA TTTCTTTGCT GTGCTGGCAATCCTCACCAT CCTCGGCGTT 3480 CTCAATGGGC TGGTTTTGCT TCCCGTGCTT TTGTCTTTCTTTGGACCATA TCCTGAGGTG 3540 TCTCCAGCCA ACGGCTTGAA CCGCCTGCCC ACACCCTCCCCTGAGCCACC CCCCAGCGTG 3600 GTCCGCTTCG CCATGCCGCC CGGCCACACG CACAGCGGGTCTGATTCCTC CGACTCGGAG 3660 TATAGTTCCC AGACGACAGT GTCAGGCCTC AGCGAGGAGCTTCGGCACTA CGAGGCCCAG 3720 CAGGGCGCGG GAGGCCCTGC CCACCAAGTG ATCGTGGAAGCCACAGAAAA CCCCGTCTTC 3780 GCCCACTCCA CTGTGGTCCA TCCCGAATCC AGGCATCACCCACCCTCGAA CCCGAAACTT 3840 CAGCCCCACC TGGACTCAGG GTCCCTGCCT CCCGGACGGCAAGGCCAGCA GCCCCGCAGG 3900 GACCCCCCCA GAAAAGGCTT GTGGCCACCC CTCTACAGACCGCGCAGAGA CGCTTTTGAA 3960 ATTTCTACTG AAGGGCATTC TGGCCCTAGC AATAGGGCCCGCTGGGGCCC TCGCGGGGCCC 4020 CGTTCTCACA ACCCTCGGAA CCCAACGTCC ACTGCCATGGGCAGCTCCGT GCCCGGCTAC 4080 TGCCAGCCCA TCACCACTGT GACGGCTTCT GCCTCCGTGACTGTCGCCGT GCACCCGCCG 4140 CCTGTCCCTG GGCCTGGGCG GAACCCCCGA GGGGGACTCTGCCCAGGCTA CCCTGAGACT 4200 GACCACGGCC TGTTTGAGGA CCCCCACGTG CCTTTCCACGTCCGGTGTGA GAGGAGGGAT 4260 TCGAAGGTGG AAGTCATTGA GCTGCAGGAC GTGGAATGCGAGGAGAGGCC CCGGGGAAGC 4320 AGCTCCAACT GAGGGTGATT AAAATCTGAA GCAAAGAGGCCAAAGATTGG AAACCCCCCA 4380 CCCCCACCTC TTTCCAGAAC TGCTTGAAGA GAACTGGTTGGAGTTATGGA AAAGATGCCC 4440 TGTGCCAGGA CAGCAGTTCA TTGTTACTGT AACCGATTGTATTATTTTGT TAAATATTTC 4500 TATAAATATT TAAGAGATGT ACACATGTGT AATATAGGAAGGAAGGATGT AAAGTGGTAT 4560 GATCTGGGCC TTCTCCACTC CTGCCCCAGA GTGTGGAGGCCACAGTGGGG CCTCTCCGTA 4620 TTTGTGCATT GGGCTCCGTG CCACAACCAA GCTTCATTAGTCTTAAATTT CAGCATATGT 4680 TGCTGCTGCT TAAATATTGT ATAATTTACT TGTATAATTCTATGCAAATA TTGCTTATGT 4740 AATAGGATTA TTTTGTAAAG GTTTCTGTTT AAAATATTTTAAATTTGCAT ATCACAACCC 4800 TGTGGTAGTA TGAAATGTTA CTGTTAACTT TCAAACACGCTATGCGTGAT AATTTTTTTC 4860 TTTAATGAGC AGATATGAAG AAAGCACGTT AATCCTGGTGGCTTCTCTAG GTGTCGTTGT 4920 GTGCGGTCCT CTTGTTTGGC TGTGCGTGTG AACACGTGTGTGAGTTCACC ATGTACTGTA 4980 CTGTGATTTT TTTTTTTGTC TTGTTTTGTT TCTCTACACTGTCTGTAACC TGTAGTAGGC 5040 TCTGACCTAT TCAGGCTGGA AAGCGTCAGG ATATCTTTTCTTCGTGCTGG TGAGGGCTGG 5100 CCCTAAACAT CCACCTAATC CTTTCAAATC AGCCCGGCAAAAGCTAAACT CTCCTCGTGT 5160 CTACGGGCAT CTGTTATGAT CATTGGCTGC CATCCAGGACCCCAATTTGT GCTTCAGGGG 5220 GATAATCTCC TTCTCTCGGA TCATTGTGAT GGATGCTGGAACCTCAGGGT ATGGAGCTCA 5280 CATCAGTTCA TCATGGTGGG TGTTAGAGAA TTCGGTGACATGCCTAGTGC TGAGCCTTGG 5340 CTGGGCCATG AGAGTCTGTA TAATAAAAAA AGCATGCAGCATGGTGCCCC TCTTTTGACC 5400 AACACACACA AGACCCCTCC CCCAACACCC CCAAATTCAAGAGTGGATGT GGCCCTGTCA 5460 CAGGTAGAAA AACCTATTTA GTTAATTCTT TCTTGGCCCACAGTCTCCCA GAAATGATGT 5520 TTTGAGTCCC TATAGTTTAA AGTCCCTCTC TTAAATGGAGCAGCTGGTTT GAGGTTTCTA 5580 AATCTGTTTG CATTTTCTTT AAAATTAAGT GGTGAGCATGCATTGTGGTG TAGAGGCACG 5640 CATTATGTAG GATAAGAGCT CCGGGGGGAT TCTTCATGCACCAGTGTTTA GGGTACGTCC 5700 TTCCTAAGTA AATCCAAACA TTGTCTCCAT CCTCCCCGTCATTAGTGCTC TTTCAATGTG 5760 ATGTGGGAAA GCAGGAGGAT GGACACACCC CACTGAAAGATGTAGGCAGG GGCAGGTCTC 5820 TCAACCAGGC ATATTTTTAA AAGTTGCTTC TGTACTGGTTCTCTTCTTTT GCTCTGAGTA 5880 GTGGGCTCCC TCATCTCGTA ACCAGAGACC AGCACATGTCAGGGAAGCAC CCAGTGTCCG 5940 CTCCCCATCC CAATCCACAC CAGCACCTTG TTACAGACAAGAAGTCAGAG GAAAGGGCCG 6000 GGTCCCTGCA GGGCTGAAGC CTAAGCTACT GTGAGGTGCTCACAAGTGGC AGCTCCTGTA 6060 ATCCCTTTTA AATTACGTGG GAATCTTAAC AGAAAGTAATGGGCCCCCAG AAATACCCAC 6120 AGCATAGGAC NTCAGACCCT GAACTCACCA CAAAATTTTAAGATGCTGAT TGGGAGCCC 6180 TTGTGGCTGC TGGATGNGTG TGTGTGTGTG TGTGTGTGCGTGCGTGCGTG TGTGTGTGTG 6240 TCTGNTGGGG ACCCTGGCCA CCCCCCTGCT GCTGTCTTGGTGCCTGTCAC CCACATGGTC 6300 TGCCATCCTA ACACCCAGCT CTGCTCAGAA AACGTCCTGCGTGGAGGAGG GATGATGCAG 6360 AATTCTGAAG TCGACTTCCC TCTGGCTCCT GGCGTGCCCTCGCTCCCTTC CTGAGCCCAC 6420 CTCGTGTTGC GCCGGAGGCT GCGCGGCCCC TGATTTCTGCATGGTGTAGA ACTTTCTCCA 6480 ATAGTCACAT TGGCAAAGGG AGAACTGGGG TGGGCGGGGGGTGGGGCTGG CAGGGAATTA 6540 GCATTTCTCT CTCTCTTTTA ATAGTTAA 6568 (2)INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1..19 (D) OTHER INFORMATION: /note= “PTC1 primer” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 2: TTGCATAACC AGCGAGTCT 19 (2)INFORMATION FOR SEQ ID NO: /note= “PTC2 primer” (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..22 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 3: CAAATGTACG AGCACTTCAA GG 22 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..18 (D) OTHERINFORMATION: /note= “W18F3 primer” (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CTGTCAAGGT GAATGGAC 18 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..19 (D) OTHER INFORMATION:/note= “W18R3 primer” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:GGGGTTATTC TGTAAAAGG 19 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 10 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 6: GAGGCTATGT 10 (2) INFORMATION FORSEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:GCCGCCATGG 10 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 6 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 8: AATAAA 6 (2) INFORMATION FOR SEQ IDNO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ATTTA 5 (2)INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1..18 (D) OTHER INFORMATION: /note= “PTCF18 primer” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 10: GAAGGCGAGC ACCCAGAC 18 (2)INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1..20 (D) OTHER INFORMATION: /note= “PTCR18 primer” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 11: TCTTTCCCTC CTCTCCCTTC 20 (2)INFORMATION FOR SEQ ID NO: /note= “PTCF22 primer” (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..18 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 12: GCTATGGAAA TGCGTCGG 18 (2) INFORMATION FOR SEQ ID NO:/note= “PTCR22 primer” (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1..20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: CAGTCCTGCTCTGTCCATCA 20 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..20 (D) OTHER INFORMATION:/note= “PTCF19 primer” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:GTGGCTGAGA GCGAAGTTTC 20 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..18 (D) OTHER INFORMATION:/note= “PTCR19 primer” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:TTCCACCCAC AGCTCCTC 18 (2) INFORMATION FOR SEQ ID NO: /note= “PTCF27primer” (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..22(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: CTATTGTGTA TCCAATGGCA GG 22(2) INFORMATION FOR SEQ ID NO: /note= “PTCR27 primer” (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..19 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 17: ATTAGTAGGT GGACGCGGC 19 (2) INFORMATION FOR SEQ ID NO:/note= “PTCF20 primer” (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1..26 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: AGAGAAATTTTTGTCTCTGC TTTTCA 26 (2) INFORMATION FOR SEQ ID NO: /note= “PTCR20primer” (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..22(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: CCTGATCCAT GTAACCTGTT TC 22(2) INFORMATION FOR SEQ ID NO: /note= “PTCF21 primer” (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..22 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 20: GCAAAAATTT CTCAGGAACA CC 22 (2) INFORMATION FOR SEQ IDNO: /note= “PTCR21 primer” (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1..22 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: TGGAACAAACAATGATAAGC AA 22 (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..20 (D) OTHER INFORMATION:/note= “PTCF15 primer” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:CCTACAAGGT GGATGCAGTG 20 (2) INFORMATION FOR SEQ ID NO: /note= “18R2primer” (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..20(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: TTTGCTCTCC ACCCTTCTGA 20 (2)INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1..21 (D) OTHER INFORMATION: /note= “11e18F primer” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 24: GTGACCTGCC TACTAATTCC C 21 (2)INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1..22 (D) OTHER INFORMATION: /note= “18R3 primer” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 25: GGCTAGCGAG GATAACGGTT TA 22 (2)INFORMATION FOR SEQ ID NO: /note= “PTCF2 primer” (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..20 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 26: GAGGCAGTGG AAACTGCTTC 20 (2) INFORMATION FOR SEQ ID NO:/note= “PTCR2 primer” (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1..20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: TTGCATAACCAGCGAGTCTG 20 (2) INFORMATION FOR SEQ ID NO: /note= “PTCF23 primer” (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..18 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 28: GTGCTGTCGA GGCTTGTG 18 (2) INFORMATION FORSEQ ID NO: /note= “PTCR23 primer” (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1..20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: ACGGACAGCA GATAAATGGC 20 (2) INFORMATION FOR SEQ ID NO: 30: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..19 (D) OTHERINFORMATION: /note= “PTCF5 primer” (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: GTGTTAGGTG CTGGTGGCA 19 (2) INFORMATION FOR SEQ ID NO: 31: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..20 (D) OTHERINFORMATION: /note= “PTCR5 primer” (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: CTTAGGAACA GAGGAAGCTG 20 (2) INFORMATION FOR SEQ ID NO: /note=“PTCF24PT primer” (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1..20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: TCTGCCACGTATCTGCTCAC 20 (2) INFORMATION FOR SEQ ID NO: /note= “CR24 primer” (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..20 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 33: CATGCTGAGA ATTGCAGGAA 20 (2) INFORMATION FORSEQ ID NO: 34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:1..19 (D) OTHER INFORMATION: /note= “PTCF16 primer” (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 34: GGCCTACACC GACACACAC 19 (2) INFORMATION FORSEQ ID NO: 35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:1..23 (D) OTHER INFORMATION: /note= “PTCR16 primer” (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 35: TTTTTTTGAA GACAGGAAGA GCC 23 (2) INFORMATIONFOR SEQ ID NO: 36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1..20 (D) OTHER INFORMATION: /note= “PTC13R primer” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 36: GTCAGCAGAC TGATTCAGGT 20 (2)INFORMATION FOR SEQ ID NO: 37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1..21 (D) OTHER INFORMATION: /note= “PTC37R primer” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 37: AAGATGAGAG TGTCCACTTC G 21 (2)INFORMATION FOR SEQ ID NO: 38: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1..20 (D) OTHER INFORMATION: /note= “PTCF14 primer” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 38: GACAGCTTCT CTTTGTCCAG 20 (2)INFORMATION FOR SEQ ID NO: 39: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1..22 (D) OTHER INFORMATION: /note= “PCTR14 primer” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 39: ACGCAAAAGA CCGAAAGGAC GA 22 (2)INFORMATION FOR SEQ ID NO: 40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1..20 (D) OTHER INFORMATION: /note= “PCTF13 primer” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 40: AGGGTCCTTC TGGCTGCGAG 20 (2)INFORMATION FOR SEQ ID NO: 41: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1..22 (D) OTHER INFORMATION: /note= “PTCR13 primer” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 41: TCAGTGCCCA GCAGCTGGAG TA 22 (2)INFORMATION FOR SEQ ID NO: 42: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1..26 (D) OTHER INFORMATION: /note= “PTCF7 primer” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 42: AACCCCATTC TCAAAGGCCT CTGTTC 26 (2)INFORMATION FOR SEQ ID NO: 43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1..23 (D) OTHER INFORMATION: /note= “PTCR7 primer” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 43: CACCTCTGTA AGTTCCCAGA CCT 23 (2)INFORMATION FOR SEQ ID NO: /note= “PTCF12 primer” (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..24 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 44: AACTGTGATG CTCTTCTACC CTGG 24 (2) INFORMATION FOR SEQ IDNO: /note= “PTCR12 primer” (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1..22 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45: AAACTTCCCGGCTGCAGAAA GA 22 (2) INFORMATION FOR SEQ ID NO: 46: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..22 (D) OTHER INFORMATION:/note= “PTCF8 primer” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:TTTGATCTGA ACCGAGGACA CC 22 (2) INFORMATION FOR SEQ ID NO: 47: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..22 (D) OTHERINFORMATION: /note= “PTCR8 primer” (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: CAAACAGAGC CAGAGGAAAT GG 22 (2) INFORMATION FOR SEQ ID NO: 48: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..23 (D) OTHERINFORMATION: /note= “PTCF11 primer” (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 48: TAGGACAGAG CTGAGCATTT ACC 23 (2) INFORMATION FOR SEQ ID NO:/note= “PTC21R primer” (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1..18 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49: TACCTGACAATGAAGTCG 18 (2) INFORMATION FOR SEQ ID NO: /note= “PTCF25 primer” (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..20 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 50: AACAGAGGCC CCTGAAAAAT 20 (2) INFORMATION FORSEQ ID NO: /note= “PTCR25 primer” (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1..18 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: GATCACTTGG TGGGCAGG 18 (2) INFORMATION FOR SEQ ID NO: 52: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..20 (D) OTHERINFORMATION: /note= “PTCF10 primer” (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 52: TCTAACCCAC CCTCACCCTT 20 (2) INFORMATION FOR SEQ ID NO: 53: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..20 (D) OTHERINFORMATION: /note= “PTC31R primer” (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 53: ATTGTTAGGG CCAGAATGCC 20 (2) INFORMATION FOR SEQ ID NO: /note=“PTCF26 primer” (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:1..19 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54: AGAAAAGGCT TGTGGCCAC 19(2) INFORMATION FOR SEQ ID NO: /note= “PTCR26 primer” (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..19 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 55: TCACCCTCAG TTGGAGCTG 19 (2) INFORMATION FOR SEQ ID NO:56: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56: GAGTACGACCCCCACACG 18 (2) INFORMATION FOR SEQ ID NO: 57: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57: GAGTACGACC CCCCACACG19 (2) INFORMATION FOR SEQ ID NO: /note= “PTCF22 primer sequence” (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 1734 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (genomic) (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:310..327 (D) OTHER INFORMATION: /note= “MuPTC1 primer sequence” (ix)FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: complement (486..505)(D) OTHER INFORMATION: /note= “MuPTCR1 primer sequence” (ix) FEATURE:(A) NAME/KEY: exon (B) LOCATION: 310..510 (D) OTHER INFORMATION: /note=“exon 1b” (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1252..1269 (D) OTHER INFORMATION: /note= “PTCF18 primer sequence” (ix)FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: complement(1384..1403) (D) OTHER INFORMATION: /note= “PTCR18 primer sequence” (ix)FEATURE: (A) NAME/KEY: exon (B) LOCATION: 1252..1440 (D) OTHERINFORMATION: /note= “exon 1” (ix) FEATURE: (A) NAME/KEY: misc_feature(B) LOCATION: 1497..1514 (ix) FEATURE: (A) NAME/KEY: misc_feature (B)LOCATION: 1732 (D) OTHER INFORMATION: /note= “end of exon 1a” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 58: ATCTTCCGCG AACTGGATGT GGGCAGCGGCGGCCAGCAGA GACCTCGGGA CCCCGCGCAA 60 TGTGGCAATG GAAGGCGCAG GGTCTGACTCCCCGGCAGCG GCCGCGGCCG CAGCGGCAGC 120 AGCGCCCGCC GTGTGAGCAG CAGCAGCGGCTGGTCTGTCA ACCGGAGCCC GAGCCCGAGC 180 AGCGAGCGGC CAGCAGCGTC CTCGCAAGCCGAGCGCCCAG GCGCGCCAGG AGCCCGCAGC 240 AGCGGCAGCA GCGCGCCGGG CCGCCCGGGAAGCCTCCGTC CCCGCGGCGG CGGCGGCGGC 300 GGCGGCAACA TGGCCTCGGC TGGTAACGCCGCCGAGCCCC AGGACCGCGG CGGCGGCGGC 360 AGCGGCTGTA TCGGTGCCCC GGGACGGCCGGCTGGAGGCG GGAGGCGCAC ACGGACGGGG 420 GGGCTGCGCC GTGCTGCCGC GCCGGACCGGGACTATCTGC ACCGGCCCAG CTACTGCGAC 480 GCCGCCTTCG CTCTGGAGCA GATTTCCAAGGTGCATTTCA GACTCTCTCC TCCCACTTTC 540 TCTTCCCTCC TCTAACTCTT TGGGATCGCCCCCGCCACAC ACAAACACAC ACACTCTCTTT 600 CCTCTCTCTC TCACACACAC ACACACATGCTCACGCTGCT GCCTCCACGA AAAGCAGCAAG 660 AGACAAATGG GGATTGAAAA ATTCAAACCCTCCCTCTGGT CCTGGGAGGA AAGGGCTGTTC 720 TGAGGTCCGC AGGGGGTGGA GGTGTGTGTGTGTGCGTGTG TGTGTGTGTA TACACACGCC 780 CTCCCTGGTG TGCCTTTTCC GGAGCACTGGAAAGCCGTCC ACGGCGGACC ACCTCAAGGGG 840 CGGCCGCGGC ACTGTCCTGC CCCGTGCCCCCTGCCCTGAA CTTCTTCCTC CTGCGCCCCCT 900 GCCCCTATTT GCAGCCTAAA CTCCTGTACGGCTGCCACAT TTCTTAACAT CTTGGAAGGGG 960 GAGCGGAGTG GAGAGAGAGC GGAGAGAGGAAGGGGGGAGG GGAGCCGAAA TAAAGGTGGT 1020 TTCCTTTTTT GCAGCCAGTT TTGTTGAGCATGAAATCTCT GCTCCATTAA AAAATTATTN 1080 TCGGAAAAAG ATATCCCCCC AGTTTTCCAGGTTTTGAGCC GCCTCTCCTT AGGGCCTGGT 1140 CGGGGGAGGA AAAGTTGTAA ACAAATTGCCACATTAAATT CGCGGTGCGA GTCTGCGGAG 1200 CTGCCGGGTT CATTGTGTNT ACGAGGCTCGCTGAAATGTG TGGAATCCAG GGAAGGCGAG 1260 CACCCAGACG GGGGCCCGCC GGGGTCGCGGCCAGCGCCGG GGAAATGCCG CGCCGGGGAG 1320 CAGCATGCGC CGGCCTGAGC CCTTCCCTTTGCACTCGGCT GTTTTTTACG TTTAACCAGA 1380 AAGGAAGGGA GAGGAGGGAA AGATCCATGTGGCTGCCCTC TTCCGATCAC AAATATTGTC 1440 GTAAGTTGCA GCTGGCTGCC CCACTTCCTAATTCAGCTCA CACAGCCTCT CCCCACGCTA 1500 TGGAAATGCG TCGGGAGTGA ACTCCGGCGGCCGCGCTCAC CACGTGGATC CCCACTTACT 1560 ACCATTCTCG GCGGGGGTCC AGTTGGGGGAACCCGCAATA TGTTGTTCCA AAGAGCGCTC 1620 GCCCCTAGCG CCCGTCCCCG AGGGTGATGGACAGAGCAGG ACTGGTTTGC TGGCTCCTGA 1680 ACCTTGGGCT CCATCGCTGG GATTACGCAGCCCCTCCCTT CTCAGCTCTG GGGT 1734 (2) INFORMATION FOR SEQ ID NO: /note=“exon 2a” (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 659 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: exon (B)LOCATION: 305..390 (ix) FEATURE: (A) NAME/KEY: misc_feature (B)LOCATION: complement (365..379) (D) OTHER INFORMATION: /note= “PTCXR1primer sequence” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59: GGATCCGTCACGTGACCCTG ACAGTTCCTG CTTATGGCGC GGCAGACCAC CCACGCCGAG 60 GGCCATGGAACTGCTTAATA GAAACAGGCT TGTAATTGTG AGTCCGCGCT GCACTCCGCCC 120 GAAAGCTTCCGGCGGCCCAG CGCGCCGGGG TTTTTACACT TTCCGTTCCT TTTGTAAAGGA 180 CGGAGGAGGAGGAGAAGAAG AAGAAGAAAA CGGAGGAGAA GAAAAAGACG ACAGGGGAGGA 240 CAAAGAGACCCGCAGCGACA AGGCAAGGGG GAGACGAGGG AAGACTGGGA GAAGACGGAAG 300 GAGCGGAGGACGAGGAAAGG GGGGCCAGGG AAAAAAAATT GATGTGAAAT CCAAGCCCGGC 360 GCTCCGAGCAGGGGTTGACG GCCGGCTATG GTNAGTGCAG CCAGCGCGGC NGCCGCCGAAC 420 GCCACCTCGCCTCTCGCGCC NTGCTCCTCG GGCGGCGCGG GGACNCTGGG ACNCGGGACCG 480 CCCCNCNCGGCGGACGGANG AGCNAGCCCC GATCGCCGGG CNGGAGGGGC GGGCCNCGCCG 540 CCNGGGCCGTGGATCCGGGT GGGCTGCGCC GCCTGGGCTC NGGANCNCTG GTCNCGCTCGA 600 TCCNCTCTCNCTCGCACNCC CGGGCCCCCG CCCCCNATGC NATCCCCTCT TGGCNGGGAGA 659 (2)INFORMATION FOR SEQ ID NO: 60: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:1296 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY:Protein (B) LOCATION: 1..1296 (D) OTHER INFORMATION: /note= “amino acidsencoded by human nevoid basal cell carcinoma syndrome (NBCCS) (PATCHED(PTC)) cDNA” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60: Met Phe Asn ProGln Leu Met Ile Gln Thr Pro Lys Glu Glu Gly Ala 1 5 10 15 Asn Val LeuThr Thr Glu Ala Leu Leu Gln His Leu Asp Ser Ala Leu 20 25 30 Gln Ala SerArg Val His Val Tyr Met Tyr Asn Arg Gln Trp Lys Leu 35 40 45 Glu His LeuCys Tyr Lys Ser Gly Glu Leu Ile Thr Glu Thr Gly Tyr 50 55 60 Met Asp GlnIle Ile Glu Tyr Leu Tyr Pro Cys Leu Ile Ile Thr Pro 65 70 75 80 Leu AspCys Phe Trp Glu Gly Ala Lys Leu Gln Ser Gly Thr Ala Tyr 85 90 95 Leu LeuGly Lys Pro Pro Leu Arg Trp Thr Asn Phe Asp Pro Leu Glu 100 105 110 PheLeu Glu Glu Leu Lys Lys Ile Asn Tyr Gln Val Asp Ser Trp Glu 115 120 125Glu Met Leu Asn Lys Ala Glu Val Gly His Gly Tyr Met Asp Arg Pro 130 135140 Cys Leu Asn Pro Ala Asp Pro Asp Cys Pro Ala Thr Ala Pro Asn Lys 145150 155 160 Asn Ser Thr Lys Pro Leu Asp Met Ala Leu Val Leu Asn Gly GlyCys 165 170 175 His Gly Leu Ser Arg Lys Tyr Met His Trp Gln Glu Glu LeuIle Val 180 185 190 Gly Gly Thr Val Lys Asn Ser Thr Gly Lys Leu Val SerAla His Ala 195 200 205 Leu Gln Thr Met Phe Gln Leu Met Thr Pro Lys GlnMet Tyr Glu His 210 215 220 Phe Lys Gly Tyr Glu Tyr Val Ser His Ile AsnTrp Asn Glu Asp Lys 225 230 235 240 Ala Ala Ala Ile Leu Glu Ala Trp GlnArg Thr Tyr Val Glu Val Val 245 250 255 His Gln Ser Val Ala Gln Asn SerThr Gln Lys Val Leu Ser Phe Thr 260 265 270 Thr Thr Thr Leu Asp Asp IleLeu Lys Ser Phe Ser Asp Val Ser Val 275 280 285 Ile Arg Val Ala Ser GlyTyr Leu Leu Met Leu Ala Tyr Ala Cys Leu 290 295 300 Thr Met Leu Arg TrpAsp Cys Ser Lys Ser Gln Gly Ala Val Gly Leu 305 310 315 320 Ala Gly ValLeu Leu Val Ala Leu Ser Val Ala Ala Gly Leu Gly Leu 325 330 335 Cys SerLeu Ile Gly Ile Ser Phe Asn Ala Ala Thr Thr Gln Val Leu 340 345 350 ProPhe Leu Ala Leu Gly Val Gly Val Asp Asp Val Phe Leu Leu Ala 355 360 365His Ala Phe Ser Glu Thr Gly Gln Asn Lys Arg Ile Pro Phe Glu Asp 370 375380 Arg Thr Gly Glu Cys Leu Lys Arg Thr Gly Ala Ser Val Ala Leu Thr 385390 395 400 Ser Ile Ser Asn Val Thr Ala Phe Phe Met Ala Ala Leu Ile ProIle 405 410 415 Pro Ala Leu Arg Ala Phe Ser Leu Gln Ala Ala Val Val ValVal Phe 420 425 430 Asn Phe Ala Met Val Leu Leu Ile Phe Pro Ala Ile LeuSer Met Asp 435 440 445 Leu Tyr Arg Arg Glu Asp Arg Arg Leu Asp Ile PheCys Cys Phe Thr 450 455 460 Ser Pro Cys Val Ser Arg Val Ile Gln Val GluPro Gln Ala Tyr Thr 465 470 475 480 Asp Thr His Asp Asn Thr Arg Tyr SerPro Pro Pro Pro Tyr Ser Ser 485 490 495 His Ser Phe Ala His Glu Thr GlnIle Thr Met Gln Ser Thr Val Gln 500 505 510 Leu Arg Thr Glu Tyr Asp ProHis Thr His Val Tyr Tyr Thr Thr Ala 515 520 525 Glu Pro Arg Ser Glu IleSer Val Gln Pro Val Thr Val Thr Gln Asp 530 535 540 Thr Leu Ser Cys GlnSer Pro Glu Ser Thr Ser Ser Thr Arg Asp Leu 545 550 555 560 Leu Ser GlnPhe Ser Asp Ser Ser Leu His Cys Leu Glu Pro Pro Cys 565 570 575 Thr LysTrp Thr Leu Ser Ser Phe Ala Glu Lys His Tyr Ala Pro Phe 580 585 590 LeuLeu Lys Pro Lys Ala Lys Val Val Val Ile Phe Leu Phe Leu Gly 595 600 605Leu Leu Gly Val Ser Leu Tyr Gly Thr Thr Arg Val Arg Asp Gly Leu 610 615620 Asp Leu Thr Asp Ile Val Pro Arg Glu Thr Arg Glu Tyr Asp Phe Ile 625630 635 640 Ala Ala Gln Phe Lys Tyr Phe Ser Phe Tyr Asn Met Tyr Ile ValThr 645 650 655 Gln Lys Ala Asp Tyr Pro Asn Ile Gln His Leu Leu Tyr AspLeu His 660 665 670 Arg Ser Phe Ser Asn Val Lys Tyr Val Met Leu Glu GluAsn Lys Gln 675 680 685 Leu Pro Lys Met Trp Leu His Tyr Phe Arg Asp TrpLeu Gln Gly Leu 690 695 700 Gln Asp Ala Phe Asp Ser Asp Trp Glu Thr GlyLys Ile Met Pro Asn 705 710 715 720 Asn Tyr Lys Asn Gly Ser Asp Asp GlyVal Leu Ala Tyr Lys Leu Leu 725 730 735 Val Gln Thr Gly Ser Arg Asp LysPro Ile Asp Ile Ser Gln Leu Thr 740 745 750 Lys Gln Arg Leu Val Asp AlaAsp Gly Ile Ile Asn Pro Ser Ala Phe 755 760 765 Tyr Ile Tyr Leu Thr AlaTrp Val Ser Asn Asp Pro Val Ala Tyr Ala 770 775 780 Ala Ser Gln Ala AsnIle Arg Pro His Arg Pro Glu Trp Val His Asp 785 790 795 800 Lys Ala AspTyr Met Pro Glu Thr Arg Leu Arg Ile Pro Ala Ala Glu 805 810 815 Pro IleGlu Tyr Ala Gln Phe Pro Phe Tyr Leu Asn Gly Leu Arg Asp 820 825 830 ThrSer Asp Phe Val Glu Ala Ile Glu Lys Val Arg Thr Ile Cys Ser 835 840 845Asn Tyr Thr Ser Leu Gly Leu Ser Ser Tyr Pro Asn Gly Tyr Pro Phe 850 855860 Leu Phe Trp Glu Gln Tyr Ile Gly Leu Arg His Trp Leu Leu Leu Phe 865870 875 880 Ile Ser Val Val Leu Ala Cys Thr Phe Leu Val Cys Ala Val PheLeu 885 890 895 Leu Asn Pro Trp Thr Ala Gly Ile Ile Val Met Val Leu AlaLeu Met 900 905 910 Thr Val Glu Leu Phe Gly Met Met Gly Leu Ile Gly IleLys Leu Ser 915 920 925 Ala Val Pro Val Val Ile Leu Ile Ala Ser Val GlyIle Gly Val Glu 930 935 940 Phe Thr Val His Val Ala Leu Ala Phe Leu ThrAla Ile Ser Asp Lys 945 950 955 960 Asn Arg Arg Ala Val Leu Ala Leu GluHis Met Phe Ala Pro Val Leu 965 970 975 Asp Gly Ala Val Ser Thr Leu LeuGly Val Leu Met Leu Ala Gly Ser 980 985 990 Glu Phe Asp Phe Ile Val ArgTyr Phe Phe Ala Val Leu Ala Ile Leu 995 1000 1005 Thr Ile Leu Gly ValLeu Asn Gly Leu Val Leu Leu Pro Val Leu Leu 1010 1015 1020 Ser Phe PheGly Pro Tyr Pro Glu Val Ser Pro Ala Asn Gly Leu Asn 1025 1030 1035 1040Arg Leu Pro Thr Pro Ser Pro Glu Pro Pro Pro Ser Val Val Arg Phe 10451050 1055 Ala Met Pro Pro Gly His Thr His Ser Gly Ser Asp Ser Ser AspSer 1060 1065 1070 Glu Tyr Ser Ser Gln Thr Thr Val Ser Gly Leu Ser GluGlu Leu Arg 1075 1080 1085 His Tyr Glu Ala Gln Gln Gly Ala Gly Gly ProAla His Gln Val Ile 1090 1095 1100 Val Glu Ala Thr Glu Asn Pro Val PheAla His Ser Thr Val Val His 1105 1110 1115 1120 Pro Glu Ser Arg His HisPro Pro Ser Asn Pro Lys Gln Gln Pro His 1125 1130 1135 Leu Asp Ser GlySer Leu Pro Pro Gly Arg Gln Gly Gln Gln Pro Arg 1140 1145 1150 Arg AspPro Pro Arg Lys Gly Leu Trp Pro Pro Leu Tyr Arg Pro Arg 1155 1160 1165Arg Asp Ala Phe Glu Ile Ser Thr Glu Gly His Ser Gly Pro Ser Asn 11701175 1180 Arg Ala Arg Trp Gly Pro Arg Gly Ala Arg Ser His Asn Pro ArgAsn 1185 1190 1195 1200 Pro Thr Ser Thr Ala Met Gly Ser Ser Val Pro GlyTyr Cys Gln Pro 1205 1210 1215 Ile Thr Thr Val Thr Ala Ser Ala Ser ValThr Val Ala Val His Pro 1220 1225 1230 Pro Pro Val Pro Gly Pro Gly ArgAsn Pro Arg Gly Gly Leu Cys Pro 1235 1240 1245 Gly Tyr Pro Glu Thr AspHis Gly Leu Phe Glu Asp Pro His Val Pro 1250 1255 1260 Phe His Val ArgCys Glu Arg Arg Asp Ser Lys Val Glu Val Ile Glu 1265 1270 1275 1280 LeuGln Asp Val Glu Cys Glu Glu Arg Pro Arg Gly Ser Ser Ser Asn 1285 12901295 (2) INFORMATION FOR SEQ ID NO: 61: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 61: ACATGTACAA CAGGCAGTGG 20 (2) INFORMATION FORSEQ ID NO: 62: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:GCAAGGAGGT TTACCTAGG 19 (2) INFORMATION FOR SEQ ID NO: 63: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 63: TGCCAAGGCT GTGGGCAAGG 20 (2)INFORMATION FOR SEQ ID NO: 64: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 64: GCTTCACCAC CTTCTTGATG 20 (2) INFORMATION FOR SEQ ID NO: 65:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65: GCTGTGACAAAGTCACATGG 20 (2) INFORMATION FOR SEQ ID NO: 66: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 66: GATGCTGCTT ACATGTCTCG 20 (2)INFORMATION FOR SEQ ID NO: 67: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:30 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 67: Ile Arg His Val Thr Leu Thr Val Pro Ala Tyr Gly Ala AlaAsp His 1 5 10 15 Pro Arg Arg Gly Pro Trp Asn Cys Leu Ile Glu Thr GlyLeu 20 25 30 (2) INFORMATION FOR SEQ ID NO: 68: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 81 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68: Val Arg Ala Ala Leu Arg ArgLys Leu Pro Ala Ala Gln Arg Ala Gly 1 5 10 15 Val Phe Thr Leu Ser ValPro Phe Val Lys Thr Glu Glu Glu Glu Lys 20 25 30 Lys Lys Lys Lys Thr GluGlu Lys Lys Lys Thr Thr Gly Glu Thr Lys 35 40 45 Arg Pro Ala Ala Thr ArgGln Gly Gly Asp Glu Gly Arg Leu Gly Glu 50 55 60 Asp Gly Gly Ala Glu AspGlu Glu Arg Gly Ala Arg Glu Lys Lys Leu 65 70 75 80 Met (2) INFORMATIONFOR SEQ ID NO: 69: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 84 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY:linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: Asn Pro Ser Pro Arg Ser Glu Gln Gly Leu Thr Ala Gly Tyr Gly Xaa 1 510 15 Cys Ser Gln Arg Gly Xaa Arg Arg Arg His Leu Ala Ser Arg Ala Xaa 2025 30 Leu Leu Gly Arg Arg Gly Asp Xaa Gly Thr Arg Asp Ala Pro Xaa Gly 3540 45 Gly Arg Xaa Ser Xaa Pro Arg Ser Pro Gly Xaa Arg Gly Gly Pro Arg 5055 60 Ala Xaa Ala Val Asp Pro Gly Gly Leu Arg Arg Leu Gly Ser Gly Xaa 6570 75 80 Leu Val Xaa Leu (2) INFORMATION FOR SEQ ID NO: 70: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70: Gly Ser Val Thr 1 (2)INFORMATION FOR SEQ ID NO: 71: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 71: Gln Phe Leu Leu Met Ala Arg Gln Thr Thr His Ala Glu GlyHis Gly 1 5 10 15 Thr Ala (2) INFORMATION FOR SEQ ID NO: 72: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72: Lys Gln Ala CysAsn Cys Glu Ser Ala Leu His Ser Ala Glu Ser Phe 1 5 10 15 Arg Arg ProSer Ala Pro Gly Phe Leu His Phe Pro Phe Leu Leu 20 25 30 (2) INFORMATIONFOR SEQ ID NO: 73: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY:linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Lys Arg Arg Arg Arg Lys 1 510 15 Arg Arg Gln Gly Arg Glu Arg Arg Thr Arg Lys Gly Gly Pro Gly Lys 2025 30 Lys Asn (2) INFORMATION FOR SEQ ID NO: 74: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74: Cys Glu Ile Gln Ala Arg AlaPro Ser Arg Gly 1 5 10 (2) INFORMATION FOR SEQ ID NO: 75: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 74 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75: Arg Pro Ala Met Val Ser AlaAla Ser Ala Ala Ala Ala Asp Ala Thr 1 5 10 15 Ser Pro Leu Ala Pro CysSer Ser Gly Gly Ala Gly Thr Leu Gly Xaa 20 25 30 Gly Thr Pro Xaa Xaa AlaAsp Gly Xaa Ala Ser Pro Asp Arg Arg Ala 35 40 45 Gly Gly Ala Gly Xaa AlaPro Gly Pro Trp Ile Arg Val Gly Cys Ala 50 55 60 Ala Trp Ala Xaa Xaa XaaTrp Ser Arg Ser 65 70 (2) INFORMATION FOR SEQ ID NO: 76: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 180 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76: Asp Pro Ser Arg Asp Pro AspSer Ser Cys Leu Trp Arg Gly Arg Pro 1 5 10 15 Pro Thr Pro Arg Ala MetGlu Leu Leu Asn Arg Asn Arg Leu Val Ile 20 25 30 Val Ser Pro Arg Cys ThrPro Pro Lys Ala Ser Gly Gly Pro Ala Arg 35 40 45 Arg Gly Phe Tyr Thr PheArg Ser Phe Cys Lys Asp Gly Gly Gly Gly 50 55 60 Glu Glu Glu Glu Glu AsnGly Gly Glu Glu Lys Asp Asp Arg Gly Asp 65 70 75 80 Ser Gly Gly Arg GlyLys Gly Gly Gln Gly Lys Lys Ile Asp Val Lys 85 90 95 Ser Lys Pro Ala LeuArg Ala Gly Val Asp Gly Arg Leu Trp Xaa Val 100 105 110 Gln Pro Ala ArgXaa Pro Pro Thr Pro Pro Arg Leu Ser Arg Xaa Ala 115 120 125 Pro Arg AlaAla Arg Gly Xaa Trp Asp Xaa Gly Arg Pro Xaa Arg Arg 130 135 140 Thr XaaGlu Xaa Ala Pro Ile Ala Gly Xaa Glu Gly Arg Ala Xaa Arg 145 150 155 160Xaa Gly Arg Gly Ser Gly Trp Ala Ala Pro Pro Gly Leu Xaa Xaa Xaa 165 170175 Gly Xaa Ala Pro 180 (2) INFORMATION FOR SEQ ID NO: 77: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 9 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77: TGTTTTAGT 9 (2)INFORMATION FOR SEQ ID NO: 78: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:9 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 78: TGTTCCAGT 9 (2) INFORMATION FOR SEQ ID NO:79: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79: CTAAACAGAGATGG 14 (2) INFORMATION FOR SEQ ID NO: 80: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 80: CTAAACAGCG TCTGGTGGAT GCAGATGG 28(2) INFORMATION FOR SEQ ID NO: 81: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 81: AATCCGCCGA TCCAGACT 18 (2) INFORMATION FORSEQ ID NO: 82: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: AGGGTGCCTG CCGTGG 16 (2) INFORMATION FOR SEQ ID NO: 83: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83: CAGGACTGTG CTCATTG17 (2) INFORMATION FOR SEQ ID NO: /note= “exon 2a” (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 85 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..85 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 84: GGAGGACGAG GAAAGGGGGG CCAGGGAAAAAAATTGATGT GAAATCCAAG CCCGCGCTCC 60 GAGCAGGGGT TGACGGCCGG CTATG 85

What is claimed is:
 1. An isolated human nucleic acid encoding a nevoidbasal cell carcinoma syndrome (NBCCS) (PTC) protein, wherein saidnucleic acid specifically hybridizes, under stringent conditions, to asecond nucleic acid consisting of a nucleic acid sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 58, and SEQ. ID NO: 59,in the presence of a human genomic library under stringent conditions.2. The isolated human nucleic acid of claim 1, wherein said isolatednucleic acid is at least 40 nucleotides in length.
 3. The nucleic acidof claim 1, wherein said isolated nucleic acid is amplified from agenomic library using any of the primer pairs provided in Table
 2. 4.The nucleic acid of claim 1, wherein said isolated nucleic acid isidentified by specific hybridization with the nucleic acids of claim 3under stringent conditions.
 5. The nucleic acid of claim 1, wherein saidnucleic acid is a nucleic acid selected from the group consisting of SEQID NO: 1, SEQ ID NO: 58, and SEQ. ID NO:
 59. 6. The nucleic acid ofclaim 1, wherein said nucleic acid includes one or more mutationscompared to a nucleic acid selected from the group consisting of SEQ IDNO: 1, SEQ ID NO: 58, and SEQ. ID NO:
 59. 7. The nucleic acid of claim6, wherein the mutation or mutations is selected from the groupconsisting of Exon 5 693 insC, Exon 17 2988 del8bp, Exon 17 3014 insA,Exon 21 3538 delG, Exon 22 G4302T, Exon 12 1711insC, Exon 12 1639insA,Exon 16 2707delC, and Intron 17 3157-2→G.
 8. The nucleic acid of claim6, wherein the mutation is a nonsense mutation.
 9. The nucleic acid ofclaim 6, wherein the mutation is a frameshift mutation.
 10. The nucleicacid of claim 9, wherein the mutation is selected from the groupconsisting of 244delCT, 217insA, 464insAC, 693insC, 804del37, 877delG,929delC, 1370del76, 1393insTGCC, 1444del6, 1497dup8, 1639insA, 1711insC,2183delTC, 2320insA, 2392delA, 2574delA, 2583delC, 2596complex,2707delC, 2748insC, 2749dup7, 2988del8bp, 3014insA, 3352delAT, and3538delG.
 11. The nucleic acid of claim 6, wherein the mutation is amissense mutation.
 12. The nucleic acid of claim 11, wherein themutation is selected from the group consisting of C391 T, G1148A,G1368A, G]525T, C2050T, C2050T, C2068T, C3015A, G3193C, and G4302T. 13.The nucleic acid of claim 6, wherein the mutation alters mRNA splicing.14. The nucleic acid of claim 13, wherein the mutation is selected fromthe group consisting of A1055-2C, 3157-2A→G, and 1493-8ins21.
 15. Thenucleic acid of claim 1, wherein the nucleic acid further comprises arecombinant vector.
 16. An isolated human nevoid basal cell carcinomasyndrome (NBCCS) (PTC) nucleic acid, wherein said nucleic acid encodes apolypeptide subsequence of at least 10 contiguous amino acid residues ofa polypeptide encoded by a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 58, and SEQ. ID NO: 59, orconservative substitutions of said polypeptide subsequence.
 17. Thenucleic acid of claim 16, wherein said polypeptide subsequence is atleast 50 amino acid residues in length.
 18. The nucleic acid of claim17, wherein said polypeptide is the polypeptide sequence encoded by anucleic acid selected from the group consisting of SEQ ID NO: 1, SEQ IDNO: 58, and SEQ. ID NO:
 59. 19. The nucleic acid of claim 16, whereinthe nucleic acid further comprises a recombinant vector.
 20. An isolatednucleic acid encoding a human nevoid basal cell carcinoma (NBCCS) (PTC)polypeptide comprising at least 10 contiguous amino acids from apolypeptide sequence encoded by a nucleic acid selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 58, and SEQ. ID NO: 59, wherein:said polypeptide, when presented as an antigen, elicits the productionof an antibody which specifically binds to a polypeptide sequenceencoded by a nucleic acid selected from the group consisting of SEQ IDNO: 1, SEQ ID NO: 58, and SEQ. ID NO: 59; and said polypeptide does notbind to antisera raised against a polypeptide encoded by a nucleic acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:58, and SEQ. ID NO: 59 which has been fully immunosorbed with apolypeptide encoded by a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 58, and SEQ. ID NO:
 59. 21. Thenucleic acid of claim 20, wherein said nucleic acid hybridizes to aclone of the human PTC gene present in a human genomic library understringent conditions.
 22. The nucleic acid of claim 23, wherein thenucleic acid further comprises a recombinant vector.
 24. An isolatedNBCCS (PTC) polypeptide, said polypeptide comprising a subsequence of atleast 10 contiguous amino acids of a polypeptide encoded by a nucleicacid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 58,and SEQ. ID NO: 59, or conservative substitutions of said polypeptidesubsequence.
 25. The polypeptide of claim 24, wherein said polypeptidecomprises a subsequence of at least 50 contiguous amino acids encoded bya nucleic acid selected from the group consisting of SEQ ID NO: 1, SEQID NO: 58, and SEQ. ID NO: 59, or conservative substitutions of saidpolypeptide subsequence.
 26. The polypeptide of claim 24, wherein saidpolypeptide is a polypeptide encoded by a nucleic acid selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 58, and SEQ. ID NO:
 59. 27.An isolated NBCCS (PTC) polypeptide, said polypeptide comprising atleast 10 contiguous amino acids from a polypeptide sequence encoded by anucleic acid selected from the group consisting of SEQ ID NO: 1, SEQ IDNO: 58, and SEQ. ID NO: 59, wherein: said polypeptide, when presented asan antigen, elicits the production of an antibody which specificallybinds to a polypeptide encoded by a nucleic acid selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 58, and SEQ. ID NO: 59; and saidpolypeptide does not bind to antisera raised against a polypeptideencoded by a nucleic acid sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 58, and SEQ. ID NO: 59 which has been fullyimmunosorbed with a polypeptide encoded by a sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 58, and SEQ. ID NO:
 59. 28.The isolated polypeptide of claim 27, wherein said polypeptide isencoded by a nucleic acid selected from the group consisting of SEQ IDNO: 1, SEQ ID NO: 58, and SEQ. ID NO:
 59. 29. An antibody whichspecifically binds a polypeptide comprising at least 10 contiguous aminoacids from a polypeptide encoded by a nucleic acid selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 58, and SEQ. ID NO: 59wherein: said polypeptide, when presented as an antigen, elicits theproduction of an antibody which specifically binds to a polypeptideencoded by a nucleic acid selected from the group consisting of SEQ IDNO: 1, SEQ ID NO: 58, and SEQ. ID NO: 59; and said polypeptide does notbind to antisera raised against a polypeptide encoded by a nucleic acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:58, and SEQ. ID NO: 59 which has been fully immunosorbed with apolypeptide encoded by a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 58, and SEQ. ID NO:
 59. 30. Theantibody of claim 29, wherein said antibody is monoclonal.
 31. Arecombinant cell expressing the antibody of claim
 29. 32. A method ofdetecting a predisposition to nevoid basal cell carcinoma syndrome(NBCCS) or to a basal cell carcinoma, said method comprising the stepsof i) providing a biological sample of said organism; and ii) detectinga human NBCCS (PTC) gene or gene product in said sample.
 33. The methodof claim 32, wherein said detecting comprises detecting the presence orabsence of a NBCCS nucleic acid.
 34. The method of claim 33, whereinsaid detecting comprises a hybridization assay.
 35. The method of claim33, wherein said detecting comprises detecting an abnormal NBCCS nucleicacid.
 36. The method of claim 35, wherein the abnormal NBCCS nucleicacid includes one or more mutations compared to a nucleic acid selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 58, and SEQ. IDNO:
 59. 37. The method of claim 36, wherein the mutation is selectedfrom the group consisting of a missense mutation, a nonsense mutation, aframeshift mutation, and a splice site mutation.
 38. The method of claim32, wherein said detecting comprises sequencing said human NBCCS gene orgene product.
 39. The method of claim 32, wherein said detectingcomprises detecting an NBCCS polypeptide.
 40. The method of claim 39,wherein said detecting comprises an immunoassay.
 41. A method oftreating nevoid basal cell carcinoma syndrome (NBCCS) in a mammal, saidmethod comprising transfecting cells of said mammal with vectorexpressing a nevoid basal cell carcinoma syndrome (NBCCS) polypeptide.42. A method of mitigating a symptom of nevoid basal cell carcinomasyndrome or a basal cell carcinoma in an organism, said methodcomprising administering to said organism a therapeutically effectivedose of a composition comprising a NBCCS (PTC) polypeptide and apharmacological excipient.
 43. A pharmacological composition comprisinga pharmaceutically acceptable carrier and a molecule selected from thegroup consisting of consisting of a vector encoding an NBCCS polypeptideor subsequence thereof, an NBCCS polypeptide or subsequence thereof, andan anti-NBCCS antibody.
 44. A kit for the detection of a NBCCS (PCT)gene or polypeptide, said kit comprising a container containing amolecule selected from the group consisting of an NBCCS polypeptide orsubsequence thereof, and an anti-NBCCS antibody.